JP2002505874A - Diagnostic and triage methods using cell activation measurements - Google Patents

Abstract

  (57) [Summary] Diagnostic methods are provided based on the use of one or more assays to assess cell activation. The assay is performed on all red or white blood cells (neutrophils) and indicates the level of cardiovascular cell activation, alone or in combination, and is central in many chronic and acute medical conditions. These results of the assay are used within the clinical framework to further test for infectious agents; antioxidant or antiadhesion treatment; postponement and optimal rescheduling of high-risk surgery; diabetes, organ transplant rejection, atheroma Classification of incidence and susceptibility to chronic disease such as venous insufficiency and its rate of progression; maximal intervention, especially in high-risk trauma cases; and supports therapeutic decisions, such as hypoactivation therapy. It is also provided as a pancreatic homogenate-derived composition containing a cell activator, which can be used as a drug screening target to identify drug candidates used for hypoactivation therapy. Also provided are methods of reducing cell activation by activating a protease inhibitor, particularly a serine protease inhibitor. A kit for performing the method is also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Some of the studies described herein were supported by USPHS Grant HL-43024. Accordingly, the government has certain rights in the subject matter disclosed herein.

RELATED APPLICATIONS US Patent Application Serial No. 09 / 038,894, filed March 11, 1998, entitled "Diagnosis and Selection Methods Using Cell Activation Measurements,"
Priority benefit to Stoughton, Geert W. Schmid-Schoenbein, Tony Hugli and Erik Kistler. If allowed, the subject matter of this application is incorporated by reference in its entirety. In the US national phase, this application is a continuation-in-part of US patent application Ser. No. 09 / 038,894.

TECHNICAL FIELD The present invention relates to the identification of cell activators and the use of cell activation as a diagnostic marker.

BACKGROUND ART Immune response and cell activation Immunity involves the recognition and processing of foreign (ie, non-self) antigenic substances present in the body. Typically, antigens are particulate matter such as cells and bacteria, large proteins, polysaccharides, and other macromolecules recognized by the immune system. Once the antigenic substance is recognized as "non-self" by the immune system, the action of specific immune cells, antibodies and the complement system induces a natural (non-specific) and / or adaptive immune response , Will be maintained.

[0005] The immune response is carried out by the immune system by natural or adaptive mechanisms, each consisting of a cell-mediated component and a humoral component. The natural mechanisms that impart innate immunity essentially mediate or participate in nonspecific immune responses, the complement system, and macrophages that react with certain bacteria, viruses, tissue damage and other antigens, Includes myeloid cells alone, such as mast cells and polymorphonuclear leukocytes (PMN).

[0006] The natural mechanisms of the immune response include phagocytic macrophages and PMNs, by which foreign substances or antigens are encapsulated and processed by their cells. In addition, macrophages can kill some foreign cells due to their cytotoxic effects. The complement system is also involved in innate immunity, which can attach to foreign substances or antigens, thereby inducing phagocytosis by macrophages and PMNs, or causing cell lysis or inflammatory effects Consists of various peptides and enzymes.

[0007] The mechanism of adaptation of the immune response is mediated by selectively responding lymphocytes (T and B cells) and antibodies. These mechanisms adapt to the environment and result in response patterns that change permanently with specific memory. Adaptive immunity is achieved by lymphocytes and antibodies alone.
Alternatively, it may be provided by the interaction of lymphocytes and antibodies with the complement system of the natural mechanism of immunity and myeloid cells. Antibodies provide the humoral component of the adaptive immune response, and T cells provide the cell-mediated component of the adaptive immune response.

[0008] The mechanism of adaptation of the immune response includes the effect of antibodies secreted by B lymphocytes (or B cells) on specific antigens, as well as on specific antigens, on B cells, on other T cells, and on macrophages. Various T lymphocytes (
Or T cells).

[0009] Lymphocytes are small cells that circulate from blood to tissues and return to the blood via the lymphatic system. There are two major subpopulations of lymphocytes called B cells and T cells. B cells and T cells are derived from the same lymphoid stem cells, B cells differentiate in the bone marrow,
T cells differentiate in the thymus. Lymphocytes have certain defined receptors that allow each cell to respond to a specific antigen. This underlies the specificity of the adaptive immune response. In addition, lymphocytes have a relatively long life span and are capable of clonal expansion upon receiving appropriate signals. This property underlies the memory context of the adaptive immune response. B cells are lymphocytes that carry the body surface of adaptive immunity. In response to recognition of specific foreign antigen, B
Cells secrete specific antibodies that bind to their specific antigens. Antibodies neutralize antigens in the case of toxins or induce phagocytosis in the case of other antigens. Antibodies can also
It is also involved in activating the complement system, which further enhances the immune response to invading antigens.

[0010] Antibodies with broad specificity for various antigens are serum globulin, which are secreted by B cells in response to recognition of the specific antigen and provide various protective responses. Antibodies can bind to and neutralize bacterial toxins, bind to the surface of viruses, bacteria or other cells recognized as "non-self," and induce phagocytosis by PMNs and macrophages. it can. In addition, antibodies can activate the complement system, which further enhances the immune response to specific antigens, and are responsible for the fluid surface of adaptive immunity.

[0011] T cells are lymphocytes that are responsible for the cell-mediated aspect of adaptive immunity. There are three main types of T cells: cytotoxic, helper, and suppressor T cells. Cytotoxic T cells detect and destroy cells infected with specific viral antigens. Helper T cells have various regulatory functions. When the helper T cell recognizes the specific antigen, it induces or enhances the antibody response to the antigen by appropriate B cells, and induces or enhances the phagocytosis of the antigen by macrophages. Suppressor T cells have the effect of suppressing the immune response directed to a particular antigen.

[0012] The cell-mediated immune response is controlled or monitored by T cells through various regulatory signaling compounds secreted by myeloid and lymphocytes. Through the secretion of these regulatory transduction compounds, T cells can regulate the proliferation and activation of other immune cells such as B cells, macrophages, PMNs, and other T cells. For example,
Foreign antigens, macrophages or other antigen presenting cells can secrete interleukin-1 (IL-1), which activates helper T cells. T cells in turn secrete certain lymphokines, including interleukin-2 (IL-2) and gamma-interferon, each of which has various regulatory effects in the cell-mediated immune response.

[0013] Lymphokines are a large family of molecules produced by T cells (and in some cases B cells), which induce clonal expansion of T cells; IL-2;
MAF, a macrophage activator that enhances many functions of macrophages, including intracellular killing and secretion of various cytotoxic factors; an activator that enhances many functions of PMNs, including phagocytosis; MIF, a macrophage migration inhibitory factor, which enriches near T cells by restricting their movement; a class produced by activated T cells that inhibits viral replication and makes these cells active in antigen binding and presentation II-interferon, which can produce a wide range of effects on many cells including induction of expression of histocompatibility molecules, activation of macrophages, inhibition of cell proliferation, induction of differentiation of some myeloid cell lines, included.

Activated macrophages and PMNs enhance the immune response as part of cell-mediated adaptive immunity and increase the production of reactive oxygen intermediates. This high production of reactive oxygen intermediates, or respiratory bursts, is known as "prime immunization." Certain lymphokines, such as gamma-interferon, trigger this respiratory burst of reactive oxygen intermediates in macrophages and. Thus, lymphokines such as γ-interferon secreted by T cells activate these macrophages and PMNs, resulting in enhanced cell-mediated immune responses.

Neutrophil Activation Thus, activation of cells is a normal physiological response essential for survival. However, inappropriate or excessive activation can also be involved in certain acute and chronic diseases. Improper stimulation of various defense strategies, including the organism itself, inflammatory cells and the immune system, often results in self-death. The first inflammatory cells to be up-regulated under these conditions are polymorphonuclear (PMN) cells or neutrophils. These cells make up 60% of the human circulating pool of leukocytes, providing a strong line of defense against invading pathogens. When activated, they produce several cytotoxic components, including free radical oxygen and proteases designed to destroy and degrade invading bacteria. When up-regulated, secreted neutrophil products also kill cells in the body and destroy tissue.

[0016] While inappropriate neutrophil activation is involved in the pathology of many disease processes, the in vivo mechanism of neutrophil activation remains relatively unknown. There are circulating plasma factors that result in up-regulation of neutrophils in hemorrhagic shock, and this up-regulation has been shown to correlate with increased mortality. The nature of this circulatory transmitter is not yet known.

History In the 1930s, the current focus on toxic circulating factors was (Menkin et al., (19
38) The American Journal of Physiology 124: 524-529; Rocha et al. (1955)
Histamine: Its Rote in Anaphylaxis and Allergy, Springfield, IL, Charles
C. Thomas.p.248; Knight et al. (1937) Br J Surg 25: 209-26; Zweifach et
al. (1957) Annals of the New York Academv of Sciences 66: 1010-1021), researchers studying shock and inflammation have adversely affected survival and have shown that neutrophil activation under these conditions can be reduced. It began when it was revealed that there were circulating factors that caused it. Several humoral factors involved in inflammation have been identified, including leukotaxin, leukocytosis-inducing factor, pyrexin (a fever-inducible polypeptide), exudin, and necrosin, each of which has a unique underlying It has inflammatory properties (Menkin (1956) Science 123: 527-534). Leukotaxin, a polypeptide recovered from inflammatory exudates, induces neutrophil chemotaxis when injected into test animals and also promotes capillary leakage (Menkin et al., (1938) The American Jou
rnal of Physiology 124: 524-529). However, it is not expected to cause damage in the cells (Menkin (1956) Science 123: 527-534), which means that neutrophil respiratory bursts are not activated by this factor. Similarly, leukotaxin has similarities with plasma derived from neutrophil chemoattractant (Petrone et al. (1980) Proc. Natl. Acad. Sci. USA 77: 1159-11).
63).

Another early factor is leukocytosis-inducing factor (LPF), which induces leukocytosis when factors present in inflammatory exudates are introduced into the circulation (
Menkin (1956) Science 123: 527-534). Leukocytosis-inducing factors have also been shown to induce hyperplasia of some hematopoietic cells, especially neutrophils (Menkin (1956) Scie
nce 123: 527-534). This factor does not cause tissue damage. However, infusion of the third inflammatory factor necrosin results in tissue damage, which is a circulating factor and has been implicated in tissue damage seen systemically during shock. Necrosin appears to be responsible for the inflammation and cell necrosis seen in many different causes of inflammation, including the damaging effects seen by ionizing radiation.

None of these factors was conclusively identified. Some, if not all, of these mediators have probably been re-identified by others, perhaps more recently, and are known by different names. Although it is important to understand and quantify the initial activation of neutrophils and the factors that bring them in vivo,
Surprisingly few studies in this area. Published reports have inconsistencies and discrepancies. It is very likely that some of the reported "factors" are in fact the same factors or are consistent with other known neutrophil activators.

[0020] Some of the most studied putative neutrophil factors are thought to be plasma components that are activated by free radical superoxide (O2-) (Petrone
et al. (1980) Proc. Natl. Acad. Sci. USA 77: 1159-1163). This factor is a lipid component non-covalently bound to serum albumin that becomes activated and becomes chemotactic when reacted with superoxide, and is thought to be produced by the xanthine / xanthine oxidase system. However, plasma exposed to superoxide in vitro was not found to stimulate degranulation or oxidative metabolism of neutrophils. Superoxide dismutase (SOD) abolishes the chemotactic response when added to plasma before exposure to superoxide, but does not abolish it when added after exposure, indicating that this activity is This indicates that it was not due to the oxide itself. However, catalase did not significantly reduce chemotactic activity when added prior to xanthine oxidase, suggesting that the chemotactic factor produced was specifically dependent on the reaction with superoxide. Is what you do. The chemotactic activity of this factor was stable at 4 ° C for 24 hours, could not be dialyzed, and was lyophilized. It is postulated that this factor may be an arachidonic acid metabolite such as 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE). This is also due to the leukotaxin identified 50 years ago (Menken et al., (1938) The Amer
ican Journal of Physiology 124: 524-529). However, the findings of Petrone et al. Overlapped with other groups that also studied this activity.

[0021] There is also a large body of research aimed at "clastogenic" factors (Emerité
t al. (1988) Ann Thorac Surg 45: 619-624; Emerit (1994) Free Radic Biol M
ed 16: 99-109; Emerit et al. (1991) Free Radic Biol Med 10: 371-377; Emeri
t et al. (1995) Free Radic Biol Med 15: 405-415; Emerit et al. (1997) Der
matologv 194: 140-146; and Emerit et al. (1996) Proc. Natl. Acad. Sci. US
.A. 93: 12799-12804), which was originally found in patients exposed to high levels of radiation and was so named for its chromosomal disruption (Emerit et al. (199
0) Methods Enzymol. 186: 555-564). Some of these factors also induce neutrophil chemiluminescence using luminol probes, possibly through the production of superoxide or other reactive intermediates (Emerit et al. (1995) Free Ra
dic Biol Med 15: 405-415). Clastogenic factors in plasma stimulate unresponsive neutrophils in vitro (Emerit et al. (1990) Methods Enzymol. 186: 555-564). Necrosin (Menkin (1956) Science 123: 527-534) may be among these factors. Clastogenic factors have been shown to be clinically produced by cardiac and lower body ischemia in as little as 38 minutes when clamping the aorta (Fabiani et at. (1993) Eur. Heart J. .: 12-17).

Not all clastogenic factors are neutrophil activators,
Also, not all neutrophil activators are clastogenic. Clastogenic factors are not produced in plasma in the absence of cells, suggesting that they are the product of cell destruction by superoxide radicals. Among the clastogenic factors, hydroxynonenal, the final product of lipid peroxidation, tumor necrosis factor-α (TNF-α) and inosine triphosphate (ITP)
Have been identified. The presence of each of these factors will depend in part on the condition being studied.
The origin of clastogenic factors in plasma of patients after cardiac ischemia is not yet known to date.

[0023] Another unknown neutrophil chemotactic factor is nourin-1 (Elgebaly et al. (1993)
Circulation 88: 1-240), found in plasma after coronary artery occlusion and thought to be produced by superoxide. It is chemotactic for neutrophils and stimulates neutrophil activation. This factor is not a product of large proteolytic cleavage, but rather a peptide composition that can be degraded by proteases (Elgebaly et al.
. (1989) J. Mol. Cell Cardiol 21: 585-593; Elgebaly et al. (1992) J Thora.
c Cardiovasc Surg 103: 952-959). Nourin-1 is water-soluble and is produced by many tissues including vascular smooth muscle, cornea, including endothelium, stomach and coronary arteries.

Recently, the discovery of neutrophil activator in plasma after the onset of ischemia has been confirmed (
Peterson et al. (1993) Ann Vasc Surg 7: 68-75; Silliman et al. (1997) Tra
nsfusion 37: 719-726; and Barry et al. (1997) Endovasc Surg 13: 381-387). These factors are seen within minutes after the onset of reperfusion and are thought to be related to superoxide production. Neutrophil activator production by plasma reperfusion was reduced by application of the PAF inhibitor WEB 2170 (Silliman
et al. (1997) Transfusion 37: 719-726) One component can be platelet activating factor (PAF) or a PAF-like substance.

Another distinct neutrophil activator set that can be separated from plasma by centrifugation at 15,000 G and from neutrophils subjected to treatment with NNaOH (Pfister et al. 1996) invest Ophthalmol Vis Sci 37: 230-237; Pfis
ter et al. (1993) invest Ophthalmol Vis Sci 34: 2297-2304) has the sequence N-acetyl-Pro-Gly-Pro (312 MW) or N-methyl-Pro-Gl
It was identified as a tripeptide having y-Pro (Pfister et al. (1995) invest
Ophthalmol Vis Sci 36: 1306-1316). These factors are long-lived and can circulate throughout the body.

Activation of cells Activated neutrophils kill free radicals, proteases and cells,
Eventually it releases many toxic substances, including those products that destroy tissue. Neutrophils also release cytokines and other inflammatory substances, which results in recruitment of additional neutrophils and activated cells, and further exacerbates inflammation and damage. In the case of a bacterial infection, this activation may be advantageous, destroying foreign pathogens otherwise harmful to the host. However, if not controlled, the effects of cell activation are considered to be extremely destructive and even lethal.

Physical stimuli such as shear stress (Moazzam et al. (1997) Proc. Natl. Acad.
Sci. USA 94: 5338-5343) and a host of chemical mediators (Ferrante (1992) Immu
Nol Ser 57: 499-521) and many other factors modulate neutrophil up-regulation. A great number of neutrophil activators of both types have been identified in vitro. Chemical agents can be broadly classified into one of two categories: receptor-mediated and non-receptor-mediated. Non-receptor-mediated neutrophil activators, such as phorbol 12-myristate 13-acetate (PMA), are generally non-specific compounds such as petroleum derivatives or detergents and are ubiquitous in number ( Wjentes et al. (1995) Semin Cell Biol 6: 357-365). Receptor-mediated factors are neutrophil-specific activators and include the bacterial peptides formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet activating factor (PAF).

Measurement of neutrophil activator has been performed in vivo as well as in vitro. Shock (Barroso-Aranda et al. (1992) Circ Shock 36: 185-190), arthritis (Dow)
ney et al. (1995) Semin Cell Biol 6: 345-356), myocardial infarction (Shandelya et al.
(1993) Circulation 87: 536-546), pancreatitis (Sandoval et al. (1996) Gastrent
erology 111: 1081-1091), and sepsis (Yoshikawa et al. (1990) Methods E
nzymol 186: 660-665) and many other medical conditions. One of the activators that has been consistently identified is PAF (Graham et al. (1994)
J Lipid Meidat Cell Signal 9: 167-182), Tumor Necrosis Factor-α (TNF-α) (Caty et al. (1990) Ann Surg 212: 694-700), Interleukins II-1 and II-8 (Ferrante (1992) Immunol Ser 57: 499-521), bradykinin (Hoffma
(1997) Microsc Res tech 37: 557-571), and LTB4 (leukotriene B4) (Letts (1987) Cardiovasc Clin 18: 101-113), and other arachidonic acid degradation products (Langholz et al. (1990) Prostaglandins Leukot Ess
ent Fatty Acids 39: 227-229).

In these cases, nothing more than a relationship with neutrophil activation was identified. Measurements are taken before and after occlusion, shock, sepsis, and other indicated injuries, and the plasma levels of the activator of interest are typically compared. Although this type of experiment provides information on the properties and potencies of various activators, the role that each mediator plays in neutrophil activation is generally unknown. After the initial stimulation of the activation of the cell population, feed-forward up-regulation of other cell types, not just neutrophils, will produce many inflammatory products. Therefore,
The presence of many activators in the circulation is generally indicative of systemic up-regulation, but not necessarily due to the single factor measured itself.

Although serine proteases are not particularly irritating to neutrophils, serine proteases have been shown to produce activators in organs that are not otherwise excitable to leukocytes. I have. Neutrophil activation by pancreatic homogenates has been shown to be inhibited by protease inhibitors. These factors are released upon shock and give the mortality and morbidity seen in various medical conditions, as well as better, apparent health. Recognition and understanding of the release mechanisms of these factors, as well as their identification, should aid in the treatment of chronic conditions in which not only shock but also inappropriate cellular up-regulation has been identified.

Thus, it is known that cells in the microcirculation are relatively dormant and are found at various activation stages. Cell activation is a normal physiological response essential for survival from infection. However, there is evidence that cardiovascular complications such as myocardial infarction, venous ulcers and ischemia / reperfusion injury are associated with activation of circulating cells. The underlying stimulus and its reasons are not yet known. Evidence that activated cells are involved in the pathogenesis of microvascular disorders confirms its origin and important key causes of activation.

Accordingly, it is an object of the present specification to identify factors responsible for activating cells.
It is also an object herein to use such factors to help understand the underlying processes and to serve as targets for the diagnosis of disease and for diagnostic intervention.

[0033] It is also an object of the present specification to provide means for improving the outcome in cardiovascular, inflammatory diseases and other diseases and conditions. It is also an object herein to provide a method of identifying a drug candidate for treatment of such diseases and conditions.

[0034]

SUMMARY OF THE INVENTION

Diagnostic methods are provided by the use of one or more assays to assess cell activation. This assay is performed on whole blood or leukocytes (neutrophils) and shows, alone or in combination, the level of cardiovascular cell activation that is central in many chronic and acute conditions. Activation of cardiovascular cells is central in many chronic and acute medical conditions, in the sense that it begins and is attributed to it. Cell activation levels are believed to be statistically correlated with disease states.

The activated state of neutrophils, endothelial cells and other inflammatory cells is important not only in conditions such as ischemia, infarction, trauma, inflammatory diseases, etc., but also for “healthy” persons. It becomes a central axis. As indicated herein, activation of such cells, particularly neutrophils, can be used both as an indicator of therapeutic outcome and as a therapeutic target. Provided herein are methods for indicating the outcome of a treatment by assessing the activation status of such cells. Cell activation may be assessed by any assay known to those of skill in the art used to measure cell activation, such as those exemplified herein. For example, activation of cells may be by the production of superoxide, such as defined by the nitroblue tetrazolium test and lucigenin-enhanced chemiluminescence, and / or by actin polymerization (such as at the level of cell activation as defined by the pseudopodia formation test). (Which is an index).

Assays are performed on whole blood, leukocytes or cell cultures and indicate the level of cardiovascular cell activation individually or in combination. These assays are intended for use with samples from human subjects, but can also be used with non-human mammalian subjects. The results of the assay are within a clinical framework to support the therapeutic decision, but are not limited to further testing for infectious agents; antioxidants or anti-adhesion treatment; postponement and optimization of high-risk surgery Re-planning; Classification of susceptibility and progression rate to chronic diseases such as diabetes, organ transplant rejection, atherogenesis, and venous insufficiency; greatest intervention, especially in high-risk trauma cases; and activity that has not been developed yet It can be used for treatment of hypoplasia.

The results of specific cell activation assays include, but are not limited to, further testing for infectious agents; antioxidant or antiadhesive treatment; postponement and optimal replanning of high-risk surgery; diabetes Classification of susceptibility to chronic diseases such as atherogenesis, and venous insufficiency and its rate of progression; maximum intervention, especially in high-risk trauma cases; and used to guide therapeutic decisions, such as hypoactivation therapy.

[0038] Also provided are treatment selection assessments and therapies, wherein the activation of cells is measured, and if it is high, a hypoactivation treatment is administered prior to further treatment. Hypotension treatments include stress management, lifestyle changes including exercise and diet, cardiac medications, administration of drugs such as aspirin, and the ethane (F
uthan)) (nafamostat mesilate, which is 6-amidino-2-naphthyl p-
Any method of reducing activity, including the administration of protease inhibitors, including guanidino-benzoate dimethanesulfonate). Although these methods and treatments are intended for use on human subjects,
It can also be used for non-human mammal subjects.

[0039] Diagnostic methods based on these assays are also provided. These assays alone,
Alternatively, one or more combinations can contribute to disease outcome and be used to support useful therapeutic decisions. The resulting diagnostics improve treatment, results and reduce cost per patient. Although these diagnostics are intended for use with samples from human subjects, they can also be used with non-human mammalian subjects.

It is also provided as a pancreatic homogenate-derived composition containing a cell activator, which can be used as a drug screening target to identify drug candidates for use in hypoactivation therapy. Compositions containing neutrophil activators found in the pancreas can activate cells both in vitro and in vivo and can be used to screen for factors that inhibit activation. In such an assay, cells, particularly cells susceptible to activation, such as PMN or endothelial cells, are contacted with the homogenate, either in the presence of the test compound, or before or after the addition of the compound. The level of activation of the cells is then assessed and a control, typically in the absence of the test compound, and / or PA
Compare to the same experiment performed either in the presence of a known activator such as F. Compounds that inhibit activation are selected as candidates for agents that can be used to block or inhibit cell activation.

The composition comprises homogenizing pancreatic tissue in a buffer, preferably at a pH of about 7 to about 8; removing particulate matter; incubating the resulting homogenate with a protease, if desired; fractionating the homogenate. And a step of selecting a fraction exhibiting a cell activating activity.

The method may also include the step of fractionating it by size and recovering components with a molecular weight of about 3 kD and / or greater than 3 kD. The homogenate (supernatant) obtained in either case can be subjected to liquid chromatography (such as high performance high pressure liquid chromatography (FPLC)) or other size or other fractionation methods. The fractions having cell activating activity are selected and combined.

[0043] Compositions made by these methods are also provided. In particular, there is also provided a composition comprising a pancreatic homogenate or an active fraction, especially an active fraction containing an active compound having a molecular weight of less than about 3 kD. These compositions are useful, for example, to identify therapeutics for diseases associated with high levels of cell activation and to identify factors that cause cell activation or diseases associated therewith.

[0044] Methods of treating diseases and conditions associated with inappropriate or chronic cell activation are also provided. In particular, treatment by administering an effective amount of a compound that reduces cell activation. Such compositions include aspirin, drugs known to reduce cell activation, novel compounds that inhibit activators in pancreatic homogenates, and enzyme inhibitors such as protease inhibitors. . Compositions containing a wide range of protease inhibitors, particularly serine protease inhibitors, and methods of treatment using the compositions are provided. In a preferred embodiment, the protease inhibitor is futan (nafamostat mesilate,
This is 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate), and is intended for treatment with a pharmaceutical composition containing an effective amount of phthalane.

Thus, in order to minimize vascular / tissue damage, futanes or similar broad protease inhibitors can be administered to shocked patients suffering from trauma or otherwise having a compromised strain (ie, Patients with activated circulating neutrophils) are provided. Administration is intended as soon as possible in the case of trauma, or immediately before surgery or invasive clinical procedures for compromised patients.

Also provided herein are drug screening assays for identifying compounds that inhibit or reduce the level of cellular activation. Assays for identifying an activator in a tissue are also provided.

[0047] Patients are tested for activation levels of cells, and the clinical trial is performed by excluding populations with high levels from the clinical population (for some standard deviations above the mean and truncation for others). Also provided is a method of increasing the probability of a patient population for: This eliminates poorly treated patients regardless of treatment,
It provides a means to better evaluate the efficacy of a clinical protocol or treatment. In other embodiments, patients or individuals with high levels can be selected for treatment in clinical trials to demonstrate the benefit of reduced cell activation therapy in disease and therapeutic outcome.

[0048] Packaging materials, labels, and protease inhibitors contained within the packaging material,
Pharmaceutical composition containing a compound effective to reduce cell activation, wherein the pharmaceutical composition is effective to reduce the level of cell activation or prevent enhanced cell activation An industrial product containing is also provided. The label indicates that the pharmaceutical composition is used to reduce the level of cell activation. The label also indicates the disease for which cell activation therapy is warranted. Packaging materials include, but are not limited to, containers, vials, blister packs, bottles, tubes,
Inhalers, pumps, bags, tubes and any means of containing suitable for delivering or storing protease inhibitors.

Kits for the diagnosis and treatment of diseases associated with cell activation include, but are not limited to, cell activation assessment means, such as tests that assess superoxide production and / or actin polymerization; Without limitation, protease inhibitors, aspirin, propranolol, heparin or coumadin,
Or means for reducing cell activation, such as other therapeutic agents that reduce activation levels. Cell activation assays or tests include the nitroblue tetrazolium test and lucigenin-enhanced chemiluminescence, and / or actin polymerization detection assays as defined by the pseudopodia formation test.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Have. All patents and publications mentioned herein are hereby incorporated by reference.

As used herein, cell activation refers to changes and interactions between circulating leukocytes including leukocytes, lining blood vessels including endothelial cells, and platelets. These changes are evidenced by increased "stickiness" of cells, changes in cell shape, generation of free radicals, and release of inflammatory mediators and enzymes. Activated cells protrude large pseudopodia and express adhesion molecules on their surface. For example, adhesion molecules and villi adhere to macrophages, and monocytes adhere to endothelium. Next, macrophages and mononuclear cells infiltrate the tissues outside the blood vessels and the development of atherosclerosis, venous insufficiency ulcers and diabetic retinopathy begins.

Cell activation is required for normal human immune defense mechanisms, but inappropriate or excessive activation includes, but is not limited to, arthritis, atherosclerosis, acute cardiovascular disease Causes, contributes to, or enhances a number of diseases, including development, Alzheimer's disease, hypertension, diabetes, venous insufficiency, autoimmune diseases, and others. Cell activation is responsible for the rejection process in organ transplantation.
And a major incentive for trends in trauma and poor outcome in high-risk surgery.

For example, LPS (lipopolysaccharide) binds to immunoglobulin M, and this complex forms C3
Activates the complement system with the release of b, which in turn is a polymorphonuclear leukocyte (PMN)
Activates monocytes, macrophages and endothelial cells. Activation of these substances is dependent on tumor necrosis factor (TNF-α), interleukin-1 (IL-1), and I
Other interleukins including L6 and IL8, platelet activating factor (P
AF), stimulates the release of several mediators of septic shock, including prostaglandins, and leukotrienes (eg, see (1991 for a more comprehensive list).
) Ann. Intern. Med. 115: 464-466). Two of these cytokines, TNF-α and IL-1, cause many physiological changes that result in septic shock.

[0054] Macrophages stimulated by LPS also release free radicals, including free radical oxygen from arachidonic acid metabolism, which can also cause extensive damage to endothelial cells. These lead to aggregation and disruption of the circulatory system, which in turn leads to hypertension, tissue damage, multiple organ failure and even death. Thus, excessive production of said free radicals has been linked to mortality associated with septic shock.

As used herein, polymorphonuclear leukocytes (PMN), polymorphonuclear neutrophil granulocytes (PMN)
A cell is a cell that has become migratory during an inflammatory event and can be stimulated by various compounds such as, for example, formylmethionyl-leucyl-phenylalanine (FMLP) or prostaglandin E (PGE1). PMN granulocytes respond to these extracellular stimuli with activation of oxygen metabolism with release of toxic oxidative metabolites. P
Excessive responses of MN granulocytes can cause painful inflammation and are accompanied by reduced levels of cyclic adenosine monophosphate (cAMP) in these granulocytes.

By “migration” with respect to PMN is meant including adhesion of the PMN to the endothelium and complete migration outward through the endothelium. Activation of leukocytes such as PMNs and monocytes and their migration to sites of inflammation are thought to occur in vivo as a result of the inflammatory response. Under normal circumstances, PMNs rarely adhere to endothelial cells, and such adhesion is considered to be the rate-limiting step in the migration process.

Activated PMN, among other mediators, causes the formation of oxygen-containing free radicals. These free radicals are produced as part of the body's defenses against the invasion of foreign organisms and their toxic products. PMN specifically generates a superoxide anion radical (O2-). These free radicals form hydrogen peroxide in the acute case through the enzyme superoxide dismutase (SOD). Excess hydrogen peroxide in the presence of iron produces a second oxygen-containing free radical, a hydroxyl free radical. In addition, activated neutrophils generate oxy radicals by stimulating the NADPH oxyreductase reaction. Release of free radical oxygen and proteases by neutrophils causes extensive damage to endothelial cells. Furthermore, the adhesion of activated neutrophils to endothelial cells leads to vascular permeability, which in turn causes massive damage associated with sepsis and septic shock.

As used herein, treatment means any method by which a condition, disease or condition of a disease or condition is ameliorated or otherwise altered to provide a benefit. Treatment also includes any of the pharmaceutical uses of the compositions herein.

As used herein, the amelioration of the symptoms of a particular disease by administration of a particular pharmaceutical composition refers to the administration of the composition, whether permanent, temporary, continuous or transient. Or any relief that can be associated with administration of the composition.

As used herein, an effective amount of a compound or composition for treating a particular disease is an amount sufficient to ameliorate, ie, alleviate the symptoms associated with the disease. Such an amount may be administered as a single dose or may be administered according to an effective dosage regimen. The amount can cure the disease, but is typically administered to ameliorate the symptoms of the disease. Typically, repeated administration is required to achieve the desired improvement in symptoms.

As used herein, reduced activation therapy (ALT) refers to any means of reducing the level of activated cells. Such measures include lifestyle and dietary changes, aspirin, pentoxifylline, Daflon 50
0 (flavonoids), anti-inflammatory drugs, pharmacotherapy such as Inderal (propanolol), heparin, coumadin, futan and other protease inhibitors.

As used herein, substantially pure refers to thin layer chromatography (TLC),
Easily measured by standard analytical methods used by those skilled in the art to assess such purity, such as gel electrophoresis, and reversed phase (hydrophobic) chromatography (ie, high pressure liquid chromatography (HPLC)). Is sufficiently homogeneous to be apparently free of detectable impurities, or can be further purified to detect the physical and chemical properties of the substance, such as enzymatic and biological activities. It is pure enough not to change. Methods for purifying compounds to produce substantially chemically pure compounds are known to those skilled in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers.
In such instances, further purification may enhance the specific activity of the compound.

As used herein, packaging material, with respect to an industrial product, means any material known to those skilled in the art that can be used to package a pharmaceutical formulation. Examples of packaging materials include
Without limitation, any of containers, vials, blister packs, bottles, tubes, inhalers, pumps, bags, tubes, and means of inclusion.

In this specification, all abbreviations for protecting groups, amino acids and other compounds are used, unless otherwise indicated, in their usual use, in approved abbreviations, or in IUPA
According to the C-IUB Commission on Biochemical Nomenclature (see (1972) Biochem. 11: 1726).

B. Cell Activation and Disease Cardiovascular cell activation is associated with acute and long-term complications (FIG. 1).
reference). Among them, cells that play major roles are endothelial cells, vascular smooth muscle, and circulating cells (red blood cells, platelets, white blood cells). These cells are encountered in a relatively dormant state, which is associated with low levels of cardiovascular complications as well as poor response following a cardiovascular attack. They can also be encountered in a more activated state associated with cardiovascular complications. This activation state involves, among other things, the production of free radicals and changes in cell morphology and adaptability, which can increase adhesion and reduce capillary flow. Such changes are part of the normal response to infection. They can, if present inappropriately or chronically, induce or contribute to conditions including myocardial infarction, hemorrhagic shock, diabetes, hypertension, and venous insufficiency.

Myocardial Infarction (MI) and Seizures (CI) Decreased flow, increased production of free radicals, and increased adhesion contribute to atherogenesis, stenosis, and eventual thrombosis via multiple mechanisms. Conceivable.
Free radicals produce oxidized low density lipoproteins (Ox-LDLs) (Steinb
erg, (1997) `` A critical look at the evidence for the oxidation of LDL in
atherogenesis, ”and enhances endothelial permeability, both of which are thought to result in monocyte infiltration and plaque formation (Lehr et al, (1992) Arter
iosclerosis and Thrombosis 12: 824-829). Decreased flow increases mechanical leukocyte adhesion to the endothelium by reducing encounter time and shear, and through activation of leukocytes associated with upregulation of expression of adhesion molecules and spontaneous morphological changes.

During and after MI or CI development, the associated ischemia and reperfusion produces free radicals and activates leukocytes (Chang et al. (1992) Biorheology 29: 549-5).
61; Grau et al. (1992) Stroke 23 (1): 33-39), increasing adhesion changes, permanently interrupting microcirculation and resulting in tissue damage (Schmid-Schonbein et al. (198
6) The American Journal of Cardiovascular Pathology 1 (1): 15-30; Welbourn
et al. Circulation Research 71 (1): 82-86; Petrasek et al. (1996) Am. J.
Physiology H1515-H1520; Jerome et al. (1993) Am. J. Physiology H479-H483
Garcia et al. (1994) Am. J. Pathology 144 (1): 188-198). Decreased brain perfusion and possibly increased blood-brain barrier permeability are also involved in progressive dementia.

Hemorrhagic shock Hemorrhagic shock was first thoroughly studied during and after World War I. This was followed by major developments in shock regulation and lethal effects of systemic hypotension (Wiggers (1995) Physiology of Shock, 1st Ed, Commonwealth Fund, N
Y, NY). In particular, Wiggers' shock model was the result of difficulties in standardizing various shock protocols. By selecting a shock protocol with a gradual systemic decrease in central blood pressure, usually to 40 to 60 mmHg, reproducible results could be obtained in anesthetized and awake animals.

[0069] Systemic ischemia and subsequent perfusion results in complications involving cell and organ damage. Tissue damage after hemorrhagic shock depends on the degree of pressure drop, the choice of loss of sensation (if appropriate), and the time of ischemia and the nature of the affected organ. Some organs, especially skeletal muscle, can survive up to 4 hours of ischemia without side effects. In other cases, such as in the visceral region and the brain, the sensitivity is higher, and a long-time low blood flow state cannot be tolerated. Organs such as the heart can only withstand brief ischemia.

From the point where not all cells have died and survival is possible,
There are probably clinically appropriate times when reperfusion is desirable. Interventions for ischemia-reperfusion, including hemorrhagic shock, must be made during the "treatment window" or until the tissue is still viable. After this, the injury is irreversible regardless of the intervention (Sussman et al. (L990) Methods Enzymol 186: 711-783). Shorter reperfusion times after ischemia result in less damage to tissue function, while longer ischemia times can result in cell death or tissue necrosis with or without reperfusion. Total occlusion of blood vessels, as opposed to low blood flow conditions, results in hypoxic cell death exclusively, not free radical interactions when reperfusion is not achieved (McCord
(1986) Adv. Free Rad Bio & Med 2: 325-345).

Hemorrhagic shock as well as ischemic conditions generally result in reduced blood flow, impaired transport of oxygen to tissues, as well as impaired waste removal. These factors impair function and ultimately kill tissue. Paradoxically, replacement of bleeding in the case of hemorrhagic shock, or re-establishment of previous blood flow to ischemic tissue, results in a phenomenon known as "reperfusion injury". This injury may be due to the re-oxygenation of the ischemic tissue and the production of free radical oxygen and other toxic substances. Free radicals, molecules with unpaired electrons, are extremely reactive and are known to cause tissue damage by disrupting cell membranes, denaturing proteins and destroying nucleic acids. Free radical oxygen affected reperfusion injury, but it is not known which free radicals are involved and where they are produced. A strong hypothesis is that hypoxia caused by hypoperfusion and subsequent oxygen exchange in ischemic tissue leads to activation and upregulation of otherwise benign enzymes and production of large amounts of free radical species It is included.

Extensive ischemia and reperfusion (I / R) in an animal is very likely to be fatal if the animal has high levels of cell activation before or simultaneously (Barr
oso-Aranda et al. (1989) Am. J. Physiological Soc H846-H852). The relationship observed between the potential for cell death and the activation of cells associated with activation in response to I / R development is that very high levels of activation following trauma or hemorrhagic shock are associated with these clinical care It suggests that it can be a particularly high risk indicator under the circumstances. For example, activation of cells probably leads to ARDS (adult respiratory distress syndrome) and MOF (multi-organ failure), where activated leukocytes block the capillary bed of the lung (ARDS) and cause the activation of other organs. Clogging capillary beds (eg, liver, kidney,
pancreas). Similarly, activation of cells due to widespread infection appears to be a major cause of death in septic shock. Therefore, based on the activation level of the patient's cells,
Vascular surgery and other open procedures can be postponed, and hypoactivation therapy can be compromised.

General health and its subclinical susceptibility In general, good perfusion is associated with health and well-being. Normal blood flow reduces the level of leukocyte activation due to shear stress (Moazzam et al. (1997) Proc. Na
tl. Acad. Sci. USA 94: 5338-5343). High levels of activation are associated with sensitivity and cardiovascular complications (Mazzoni et al. (1996) Cardiovascular Research 32: 7
09-719). Thus, asymptomatic susceptibility to health is closely related to inappropriate activation levels, and it is reasonable to predict that asymptomatic diseases, such as asymptomatic infections, are caused by increased activation levels. is there.

Diabetes, hypertension and venous insufficiency Activation of cells of the cardiovascular system, such as leukocytes or endothelial cells, is associated with diabetic retinopathy (Schrod
er et al. (1991) Am. J. Pathology 1398 (1): 81-100) and venous insufficiency disease (Edwa
rds et al. (1994) "White blood cell distribution in chronic venous insuf
ficienct ", Chapter 7 of Microcirculation in Venous Disease, Smith, ED.)
Have the potential to accelerate the onset of diabetes by free radical damage to pancreatic B cells mediated by or independent of hyperglycemia (Schroder et al. (1991) Am. Pathology 139 (1): 81-100).

Activation levels, especially free radicals and living lipids, can also mediate hypertension (Sagar et al. (1992) Molecular and Cellular Biochemistry 111: 10).
3-108; Shen et al. (1995) Biochem.Cell Biol. 73: 491-500; Schmid-Schonbe
in et al. (1991) Biochem. Cell Biol 17 (3): 323-330).

C. Cell Activation Diagnostics and Therapeutic Points The use of cell activation for diagnostic and therapeutic intervention is illustrated in FIG. 2, which is a method for evaluating the treatment options provided herein. It shows the paradigm of. Activation is the center of disease outcome, trauma outcome, and long-term systemic health. Identification of apparently healthy individuals with elevated levels of activated cells allows for early identification of dangerous individuals, as well as early intervention in chronic and acute illnesses. Activation levels are measured in apparently healthy patients, as shown in FIG. If it is low, treatment or lifestyle changes may not be recommended. If the level is high (greater than about 50%, more likely greater than 20%, or standard deviation above mean), the presence or absence of subclinical infection or other cell activating conditions Perform a test to check If those tests are negative, lifestyle and diet should be considered and improved if necessary. If the diet is good, the lifestyle is generally good, and there is no stress, hypoactive treatment can begin.

Testing cell activation levels prior to surgery, particularly elective surgery, can be used to assess the potential for surgical complications and organ transplant rejection.
Surgery should be postponed if high levels of cell activation are seen that are not the result of infection. Hypoactivation treatment is considered. Similarly, in unstable angina, the level of cell activation is indicative of the risk of cardiovascular failure. Therefore, if the level is high, hypoactivation therapy and / or more aggressive therapy should be performed. In a trauma situation, the level of cellular activity may aid in selecting a treatment protocol and its timing. High levels of activation are associated with ARDS and MOF in the emergency room
Is related to Hypoactivation treatment must reduce that risk.

Thus, generally, if a high level of cell activation is observed, a reduced activation treatment should be administered prior to further treatment. Hypotension treatments include administration of known medicaments such as aspirin and cardiovascular drugs, extracorporeal blood treatments such as filtration techniques, and other such treatments. As indicated herein, a protease inhibitor, particularly a serine protease such as futan, can be administered. It is also contemplated herein that the compounds identified using the methods herein for such identification are administered.

Cell activation is statistically correlated with disease state. It is considered elevated if it goes beyond the normal range that can be established by sampling the "healthy" person and averaging it. In particular, individuals who have activated cells 10% or 20% higher or at some standard deviation above the mean are considered candidates for hypoactivation therapy.

Cell Activation Detection Test In practicing the present method, one or more cell activation tests are performed. These tests are discussed and illustrated in further detail below, and include tests that evaluate indicators of activation, such as morphological changes and free radical production. For example, morphological changes in cells may be quantified by direct microscopic observation with or without fluorescent staining of F-actin filaments present in pseudopodia, or by a fluorescence activated cell sorting technique. The production of superoxide anion is luminol, which reacts quantitatively with it,
It can be detected and quantified using chemiluminescence producing reagents such as isolminal and lucigenin. Free radicals can be evaluated by NBT (nitro blue tetrazolium). Adhesion can be assessed by various immunoassays that detect surface adhesion molecules such as CD11, CD18 and L-selectin and others. Other indicators of activation include the expression of certain factors such as interleukins and TNF-α,
This can be measured by known immunoassays.

Activation can also be assessed by taking the patient's plasma and examining whether it activates cells, such as endothelial cell culture. Plasma can be tested for clastogenic activity by standard methods. Although there is a high correlation between the measurements of the various cell activation assays, there are likely to be various combinations of indicators that will give the most information in any given situation. For example, plasma activator levels may be high, but the activated neutrophil content in the circulation is low due to the retention of activated cells in the microcirculation. Also, genetic, age, and environmental differences between patients complicate the interpretation of the assay. Clinical trials should be used to statistically correlate cell activation measurements with disease outcomes, to find formulations that are invariant to patient differences, and to provide good predictive methods and activation in a variety of contexts. Being prepared to establish reduction therapy. Cell activation and measurement of circulating plasma factors also serve as an effective tool to assess the effectiveness of new interventions prior to conducting full clinical trials. This may reject drug candidates, or may increase the patient population's probability of a more favorable response to the candidate drug.

D. Pancreatic Neutrophil Activating Compounds Factors circulating in rat shock plasma activate unresponsive neutrophils (eg,
Mazzoni et al. (1996) Cardiovasc Res. 32: 709-719; Barroso-Aranda et at.
(1989) Am J Physiol: H846-852; Barroso-Aranda et al. (1992) Circ Shock 36
: 185-190; Barroso-Aranda et al. (1989) Am J Physiol: H415-421; and Shen e
tal. (1990) Circulatory Shock. 31: 343-344). These studies also show that the level of neutrophil activation in vitro induced by plasma collected prior to the shock protocol conversely corresponds to the animal's survival in shock,
This leads to the recognition of "preactivation". Pre-activated neutrophil levels (activation of "resting" neutrophils before shock) correlate with lipid peroxide production in hemorrhagic shock experiments. The results of lipid peroxidation over time are also consistent with those of plasma peroxide in hemorrhagic shock, but it is not clear how the two measurements are related. Oxidation of lipid membranes can lead to the increase seen in plasma peroxide.

[0083] Hemorrhagic shock is a generalized seizure and no information is given on the possible origin of these factors. A rat splanchnic artery occlusion (SAO) shock model was studied. Previous work (Lefter et al. (1970) Circ Res 26: 59-
69) show that myocardial inhibitory factor (MDF) is produced during hemorrhagic shock. MDF production is enhanced in the SAO shock model due to more complete ischemia and subsequent autolysis of the pancreas than hemorrhagic shock. It has been hypothesized that MDF is co-located, or perhaps simultaneously, with the in vivo neutrophil activator measured in hemorrhagic shock. SAO shock has been shown to result in the release of plasma factors that activate neutrophils in vitro, which implies a site of production of neutrophil-activating plasma factors as a visceral region.

The presence of plasma-induced neutrophil activators after SAO shock indicated that the visceral region was a potential site of formation of these factors. To study this possibility, rat homogenates were made of this visceral organ as well as other representative viscera. Homogenize the liver, intestine, heart, spleen, pancreas, adrenal gland and kidney tissue and allow the homogenate to be stimulated at 38 ° C. at 2 ° C. The neutrophil activation properties were measured in vitro before and after the 0.5 hour incubation. Of the tissues measured, only pancreatic homogenate stimulated neutrophils to a significant degree. Neutrophil activator was also found in porcine pancreatic homogenate, indicating that the pancreatic activator is not species-specific. Incubated pancreatic homogenates activated neutrophils to a greater extent than unincubated samples, but unincubated pancreatic homogenates were still significantly irritating to unresponsive neutrophils. These findings demonstrate that of the organs measured, the pancreas is the only tissue that contains appreciable amounts of neutrophil activator as measured in vitro, and that such factor has already been preformed. It suggests that. In contrast, MDF activity is not present in unincubated pancreatic homogenates,
This indicates that its production requires an enzymatic step. The enhanced neutrophil activation seen in the incubated homogenates reflects the need for lysosomal degradation or cell lysis for maximal activator release.

The activators found in the pancreas are not considered to be essentially proteases since direct incubation of neutrophils with trypsin and chymotrypsin does not activate neutrophils. Preliminary isolation of pancreatic homogenates indicates that there are many activators produced in the pancreas, including low molecular weight (<3 kD) activators that may contain platelet activating factor-like (PAF-like) substances It is suggested that Further work must be done to identify the final properties of these activators.

[0086] Further experiments to identify the origin of these factors were designed to determine the ability of rat tissue homogenates to activate neutrophils in vitro. Of the tissues tested, only the pancreas has the ability to activate unresponsive neutrophils in vitro. As demonstrated herein, the pancreas is considered to be a source of circulating plasma factors that activate unresponsive neutrophils in hemorrhagic shock, and also inhibit myocardial depression, multiple organ failure and It is thought to cause death. This pancreatic cell activator appears to be of low molecular weight (<3000 Da).

As shown herein, cell activators are released from these other homogenates when incubated with homogenates of other organs, pancreatic homogenate supernatants, and trypsin and chymotrypsin. Serine protease inhibitors, such as futan, inhibit the production of cell activators in in vitro experiments and reduce the systemic response in vivo. These and other observations suggest that the pancreas is a source of endogenous proteases that cleave active fragments from pancreatic and non-pancreatic proteins.

The following experiments showed that other tissues could be excitatory to neutrophils by the addition of limited concentrations of pancreatic homogenate or serine protease. Protease inhibitors, especially serine protease inhibitors futan (nafamostat mesilate, non-peptidyl low molecular weight protease inhibitor 6-amidino-2-naphthy-p-buanidinobenzoate dimethanesulfonate; Fuji e
(1981) BioChim. Biophys. Acta 661: 342) reduces neutrophil activation in vitro and reduces mortality in animals receiving either SAO shock or pancreatic homogenate infusion.

Experimental Results—Summary The results of experiments performed to identify and characterize neutrophil activators in vivo are summarized in each example. Some key points are highlighted here for review.

Provided herein are compositions containing factors that activate cells, including neutrophils, and partially purified pancreatic homogenates. The composition has low molecular weight components (<3 kD)
As well as possibly higher molecular weight factors. This homogenate and its fraction are powerful activators. Homogenates may be used as screening agents to identify inhibitors of cell activation (see below). The identification of its unique component allows the production of antibodies for diagnostic purposes, as a target for drug design, and as a screening agent to develop specific hypoactive agents.

Several protease inhibitors have been studied for their ability to inhibit neutrophil activation induced by pancreatic homogenate. Serine protease inhibitors were able to successfully alter the extent of inhibition of neutrophil activation by pancreatic homogenates in vitro. Of these inhibitors, the serine protease inhibitor futan (nafamostat mesilate) was found to be the most effective. Experiments with neutrophils washed with unbound futan show similar inhibition as experiments with futan added directly to the homogenate, indicating that the mechanism of inhibition of neutrophil activation of futan is associated with neutrophil membranes. And suggests that it is not necessarily directed to a homogenate. This conclusion is based on the observation that high concentrations of the major pancreatic proteases, trypsin and chymotrypsin (alone and in combination with their precursors, trypsinogen and chymotrypsinogen), do not result in neutrophil activation in vitro. Can be further strengthened. further,
Neutrophil activation was also observed in the pancreatic homogenate from which a factor of 3 kD or more had been removed by filtration, indicating that at least some of the neutrophil activating factors in the pancreatic homogenate were any of the known proteases (approximately 20 to It is much smaller than 300 kD), suggesting that it is of low molecular weight. Activation was also retained in high molecular weight pancreatic samples, but does this activity mean another factor, a low molecular weight factor bound to large proteins, or simply low retention retained after filtration? Whether it implies a molecular weight factor is not clear at this time.

As a series of control experiments, a partially activating concentration of pancreatic homogenate was added to other organ homogenates, ie, liver, spleen, intestine, and heart, which have so far shown little ability to activate neutrophils. added. Surprisingly, incubating these tissues with low concentrations of pancreatic homogenate gave them the ability to potently activate neutrophils. Further experiments demonstrated that the neutrophil activating power of a previously inactive organ homogenate could be doubled by the addition of the pancreatic proteases chymotrypsin or trypsin. As mentioned above, neither chymotrypsin nor trypsin essentially activate neutrophils in vitro, indicating that heart, liver, spleen and intestinal homogenates are non-stimulatory for neutrophils. I have. However, the addition of protease provided these tissues with the ability to activate neutrophils in vitro. The mechanism that supports this activation is not yet clear. Platelet activating factor (PAF) has been reported to be activated by endothelial cells incubated with thrombin and chymotrypsin and cathepsin G and can be inhibited by the addition of protease inhibitors. Thus, this ability to stimulate neutrophils by the homogenate incubated with the serine protease is linked to their ability to synthesize PAF. Preliminary results suggest that the low molecular weight fraction corresponding to PAF (<3 kD) has little activity, and that PAF inhibitors are not effective in reducing homogenate-mediated neutrophil activation Was.

To correlate the in vitro results obtained with pancreatic homogenates on neutrophil activation with their inhibition by serine proteases to the in vivo state,
The SAO shock experiment was repeated using pretreatment with futan. After determining the optimal concentration and infusion parameters, pretreatment with futan for 60 minutes was found to alleviate the decrease in mean arterial pressure (MAP) seen after reperfusion (releasing the clasp) with SAO shock. Mortality was significantly reduced and plasma peroxide production levels were significantly lower than saline-treated control rats. The mechanism of protection by futan is due to several factors, including reduced neutrophil activation in vivo, stabilization of pancreatic liposomes and atrial membranes, and a general increase in protective circulating antiprotease screens. Conceivable.

Close injection of reperfusion MAP in SAO shock by injection of filtered pancreatic homogenate into animals resulted in increased circulating peroxide production and immediate death seen in SAO shock. Pretreatment of the animals with futan increased MAP in response to injection of pancreatic homogenate, eliminating the mortality seen in untreated animals. In addition, as a result of the injection of the low molecular weight component of the pancreatic homogenate, MAP was significantly reduced. However, the blood pressure of these animals recovered after a blood pressure reduction time of about 10 minutes and the animals did not shock at the given concentration (up to 30% of low molecular weight components from one pancreas / animal). This decrease in blood pressure is probably also a low molecular weight substance, and therefore M, which should be present in the low molecular weight fraction with the neutrophil activator.
DF.

In vivo microscopy of rat mesentery infused with filtered pancreatic homogenate showed a significant increase in neutrophil activation and vasoconstriction, concluding that neutrophils as well as other cell types Also demonstrated an in vivo role of pancreatic homogenate in activation. Endothelial and parenchymal cell death did not increase significantly in these experiments, indicating that pancreatic homogenate was not a source of neutrophil activator but was directly cytotoxic to tissues. It suggests.

A variety of methodologies have been used to isolate and identify neutrophil activators found in the pancreas. In an attempt to purify neutrophil activating activity, low molecular weight pancreatic homogenates (<3k
D) was subjected to high performance high pressure liquid chromatography (FPLC). Fractions were measured for their neutrophil activation ability and the "activated" fraction was further analyzed by high pressure liquid chromatography (HPLC) (reverse phase (hydrophobic) chromatography).
And purified. The HPLC fraction was then analyzed for neutrophil stimulating activity, and the "activated" fraction was measured by a MALDI mass spectrometer. The known peptide sequence and low molecular weight transmitter were analyzed by computer, and the measured molecular weights were compared. Based on these results, several known activators could be eliminated as pancreatic low molecular weight factors. Among those factors excluded were PAFs (containing either 16 or 18 carbon alkyl groups.
Also known as "true PAF"), fMLP, bradykinin, angiotensin II, and all known cytokines. Although some peptide sequences have molecular weights equivalent to those measured by mass spectrometry, none of them have been reported to retain any neutrophil stimulating activity. The results obtained from the frozen pancreatic homogenate are contradictory, but appear to have reduced low molecular weight activity. Lyophilization of the low molecular weight pancreatic homogenate was not as successful as dialysis, and preliminary attempts to extract the activator into methanol or chloroform were similarly largely unsuccessful. The activity of the whole pancreatic homogenate, as well as the low molecular weight, is relatively stable at 4 ° C., and the whole molecular weight homogenate can be stored for at least one week. In contrast, factors produced by application of proteases to homogenates of other organs are relatively unstable, with half-lives on the order of about 8 hours.

1. Tissue homogenates incubated with serine proteases contain factors that activate PMN in vitro. Visceral artery occlusion (SAO) shock results in up-regulation of neutrophil (PMN) activation levels, Is measured by pseudopodia formation of donor PMN plasma exposed to shock. Except for pancreatic homogenates, homogenates consisting of rat peritoneal organs do not significantly activate isolated unresponsive PMNs. Pancreatic activation can be inhibited in vitro by the addition of a serine protease inhibitor.

The addition of a foreign protease results in activation of other tissues. Randomly selected rats were weighed, anesthetized and catheterized. An abdominal wall incision was made and whole blood was collected from the animals. Organs were immediately removed in 0.25M sucrose solution and then homogenized in 1: 9 (w / v) Krebs-Henseleit solution.

The organs collected included spleen, adjacent small intestine, pancreas, heart and liver. Aliquots of each sample were mixed with serine protease, chymotrypsin, and trypsin. The suspension was incubated at 38 ° C. for 2.5 hours and the activation of PMN was measured. The results were due to pancreatic homogenate in activation of PMN (p <
0.01), as well as (p <0.01) from tissue homogenates incubated with protease. Activation from homogenates of control organs other than the pancreas was not enhanced. These results indicate that tissue homogenates incubated with serine proteases contain factors that activate PMN in vitro. The pancreas may serve as an intrinsic source of PMN activator.

2. Inhibition of Activation by Futan in Vivo Plasma factors that occur during splanchnic artery occlusion (SAO) shock result in up-regulation of leukocytes, as measured by nitroblue tetrazolium (NBT) or pseudopod activation Is done. Homogenates from the pancreas will activate unresponsive neutrophils, while those from other tissues will hardly activate unresponsive neutrophils by similar tests. This activation is based on the activation of futan (nafamostat mesilate: a non-peptidic low molecular weight protease inhibitor 6-amidino-2-
Naphthyl-p-guanidinobenzoate dimethanesulfonate; Fuji et al. (1
981) Some inhibition in vitro by serine protease inhibitors such as Biochim. Biophys. Acta 661: 342).

To demonstrate that activation was inhibited in vivo, rats were anesthetized and blood pressure was monitored (MAP). Futhan was injected at a rate of 3.3 mg / kg wt / hr. After 1 hour of pretreatment, the upper mesenteric and peritoneal arteries were clamped for 90 minutes, at which point the clamp was removed. 60 minutes after reperfusion or M
Animals were observed for survival until the AP dropped below 30 mmHg. Plasma peroxide concentration was measured using electrode technology.

The results show a significant difference (p <0.005) in MAP after reperfusion between futan-treated and untreated animals and a significant increase in survival of futan-treated animals compared to controls (P <0). .001). Also, peroxide levels in futanized SAO shock plasma were significantly lower than in controls (p <0
.05). This result indicates that pretreatment with a serine protease inhibitor mitigates SAO shock and that this protection may be somewhat due to the ability of the protease inhibitor to limit the level of circulating activators during shock. .

E. Cell Activation Assay The rate of free radical production in whole blood was determined by phenol red (Pick et al. (198
0) J. Immunol. Methods 38: 161-170) or other color formers (US Pat. No. 5,5).
18,891). Intracellular radical production is determined by nitroblue tetrazolium (NBT) reduction or chemical fermentation assay (Cheung et al. (1984)
Aust. J. Expt. Biol. Med. Sci. 62: 403). Radical production in whole blood or plasma may be measured electrochemically, and mRNA expression of specific genes may be quantified using, for example, Northern blots or DNA microassays.

The expression of adhesion molecules such as CD11b, CD18 and L-selectin can be quantified by flow cytometry, while cytokines and chemokines such as interleukins and TNF-a can be quantified by immunoassay.

The morphological changes of the cells can be quantified by direct microscopic observation with or without fluorescent staining of F-actin filaments present in pseudopodia, or by fluorescence activated cell sorting.

Blood plasma is known to carry cell activators that respond to specific events. MI (Chang et al. (1992) Biorheology 29: 549-561) and hemorrhagic shock (Elgebaly et al. (1992) J. of Thoracic and Cardiovascular Surgery 1)
03 (5): 952-959; Paterson et al. (1993) Am.Vasc. Surg. 7 (1): 68-75; Barroso
-Aranda et al. (1995) Plasma from I / R episodes, including J. Cardiov Pharmacology 25 (Suppl 2): S23-S29), activate neutrophils, and Plasma from blood is also activated (Pitzer et al. (1996) Biorheology 33 (1): 4
5-58). Patient blood samples could be applied to standard donor cells and the donor cell response was used as a measure of circulatory activator efficacy in the patient's blood.

F. Therapeutic Framework A test for activation would be meaningless without a constitutive response to the information obtained in the study. The response can take the form of lifestyle or dietary adjustments such as increased exercise, reduced fat intake, postponement of scheduled surgery, antioxidant and hypoactive drug therapy, or antagonists to circulating plasma factors. An example of a treatment decision diagram is shown in FIG.

Highly active but apparently healthy patients are advised to adjust their lifestyle and diet or use antioxidants (Stephens et al. (1996 The Lancet 3
47: 781-786) or aspirin (Ridker et al. (1997) New England J. Medi.
A relatively harmless activation reduction therapy such as cine 336 (14): 973-979) could be administered. Patients in high-risk surgery with high activation levels can postpone surgery or receive hypoactivation therapy. An example of an existing protocol is Centocor's platelet aggregation blocker (Leopro) given for high-risk angiogenesis. So far, patients with unstable angina have been treated with anti-adhesion agents for angioplasty for drug therapy to reduce activation or for bypass surgery from no treatment (Husten, "Platelet receptor blockers effective for unstable angina ”Int
ernal Med. World Report, May 15, 1997). These choices can depend on the degree of cell activation observed. Unstable angina has been shown, for example, to be associated with altered expression of CD11b and L-selectin in neutrophils (Ott et al. (1996) Circulation 94 (6): 1239-1246).

In some cases, the high activation level is in response to infection. If the infection is asymptomatic, activation tests may provide a clue to its presence. If the infection is apparent for other reasons, treating it or waiting for it to subside is the first step in responding to hyperactivation under nonclinical attention. Activation by asymptomatic infection can be suppressed by antibiotics.

Finally, the consequences of trauma and sepsis are indicated by the presence of circulating plasma factors and by the extreme levels of activation observed, so that the choice of maximal intervention would be more reasonably selected . Serine protease inhibitors, such as futan, are effective in vivo in animal models for hemorrhagic shock and clearly prevent the action of factors that occur in the pancreas. Thus, existing protease inhibitors should be useful in treating septic hemorrhagic shock and could also be used as drug targets.

The therapeutic target will preferably be either an agent such as one that is released from the pancreas and activates the cell, or a protease that participates in the activation.

Treatment with Protease Inhibitors Leakage of pancreatic proteases and other factors into the bloodstream, or excessive activation without proper endogenous inhibitor control in the pancreas has life-threatening consequences. It is generally known that pancreatic damage is fatal, and it is important to inhibit protease action at the level of leukocytes in order to minimize post-ischemic damage. Taken together, drugs that can be effective in preventing the generation of cell activators from tissues, even proteases that play this role, should be therapeutically useful and have many clinical applications. This type of intervention has the potential for early intervention in the transmitter / activator cascade and is particularly effective in minimizing post-injury phenomena.

[0113] Thus, methods for treating diseases and conditions associated with inappropriate or chronic cell activation are provided. In particular, treatment is provided by administration of an effective amount of a broad range of protease inhibitors, particularly serine protease inhibitors. In a preferred embodiment, the protease inhibitor is phthalane (nafamostat mesilate, which is 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate), and is treated with a pharmaceutical composition containing an effective amount of phthalane. Is intended.

Shock patients who suffer from trauma or otherwise have a susceptible strain using a protease inhibitor such as futan or similar broad protease inhibitors to minimize vascular / tissue damage (Ie, patients with activated circulating neutrophils). In the case of trauma, administration is intended as soon as possible or, in the case of compromised patients, immediately before surgery or open clinical procedures. The dosage is (with respect to futan) approximately 0.001 to 1 mg / ml, preferably about 0.005, based on blood volume by any suitable method, including intravenous, intramuscular, oral and parenteral administration. To 0.05 mg / ml, more preferably about 0.01 mg / ml. Thus, the average adult receives about 50 mg of phthalane per dose. Since the compound is a low molecular weight drug and can be excreted relatively quickly, the frequency of treatment may be as low as 6 to 8 hours during acute episodes and as small as a single dose for surgical patients. The exact dosage of a particular inhibitor can be determined empirically and will depend on the particular disease being treated and the desired result.

Attention should be paid to monitoring for bleeding and damage to the host's humoral defense mechanisms. Should be. Futan is relatively non-toxic in humans and is well tolerated.

G. Formulation and Administration of Active Compounds and Compositions Compounds such as, but not limited to, serine protease inhibitors and protease inhibitors, including futan, and compositions containing such proteases. Is provided. These compounds can be derivatized as the corresponding salts, esters, acids, bases, solvates, hydrates and prodrugs. The concentration of the compound in the formulation is effective to deliver an amount that reduces or inhibits cell activation upon administration. Typically, the compositions are formulated for single dose administration. To formulate the composition, the compound or mixture thereof is dissolved, suspended, dispersed, or otherwise dissolved in the vehicle of choice at an effective concentration so as to reduce or ameliorate the condition treated in the chosen vehicle. Mix. Suitable pharmaceutical carriers or vehicles for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for a particular mode of administration.

In addition, these compounds may be formulated as the sole pharmaceutically active ingredient in the composition, or may be combined with other active ingredients. A liposome suspension containing a liposome targeting a tissue may also be suitable as a pharmaceutically acceptable carrier. These can be manufactured according to methods known to those skilled in the art. For example, a liposome preparation may be prepared as described in US Pat. No. 4,522,811.

The active compound is contained in a pharmaceutically acceptable carrier in an amount sufficient to produce a therapeutically useful effect without undesirable side effects for the patient being treated. A therapeutically effective concentration may be determined empirically by testing the compound in known in vitro and in vivo systems, such as the assays provided herein.

The concentration of the active compound in the pharmaceutical composition will depend on the rate of adsorption, inactivation and excretion of the active compound, the pharmacological properties of the compound, the dosage regimen and dosage, and other factors known to those skilled in the art. I do.

Typically, a therapeutically effective amount will be about 0.001 to 1 mg / ml, preferably about 0.005 to 0.05 mg / ml, more preferably about 0.001 to 1 mg / ml of blood volume. 0.01 mg / ml. Pharmaceutical unit dosage forms may contain from about 1 mg to about 1000 mg and preferably from about 10 to about 500 mg, more preferably from about 25 to 75 mg, per unit dosage form, of the essential active ingredient or combination of essential ingredients.
Is prepared to provide

The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the exact dosage and duration of treatment will be correlated to the disease being treated and may be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. In addition, the particular dosage regimen should be adjusted over time according to the individual needs and professional judgment of the person administering or directing the administration for any particular patient, and It is also to be understood that the concentration ranges set forth in the specification are merely exemplary, and are not intended to limit the scope or use of the claimed compositions.

Preferred pharmaceutically acceptable derivatives include acid, salt, ester, hydrate, solvate and prodrug forms. Typically, the derivative is selected to have better pharmacokinetic properties than the corresponding neutral compound.

Accordingly, an effective concentration or amount of one or more of the compounds provided herein or a pharmaceutically acceptable derivative thereof is mixed with a pharmaceutical carrier or vehicle suitable for systemic, topical or local administration. To form a pharmaceutical composition. The compound is included in an amount effective to ameliorate or treat the disease for which treatment is intended. The concentration of the active compound in the composition will depend on the adsorption, inactivation, elimination rate, dosage regimen, dosage, particular formulation of the active compound, the particular formulation and other factors known to those skilled in the art.

The compositions are intended to be administered by a suitable route, and include oral, parenteral, rectal and topically and locally depending on the disease to be treated. For oral administration, capsules and tablets are presently preferred. Liquid, semi-liquid or solid compounds are formulated to be suitable for each route of administration. Preferred modes of administration include parenteral and oral modes of administration.

Solutions or suspensions for parenteral, intradermal, subcutaneous or topical application include any of the following components: water for injection, saline solution, fixed oils, polyethylene glycol, glycerin. Sterile excipients such as propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol and methyl paraben; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); Buffers such as citrate and phosphate; and osmotic agents such as sodium chloride or glucose. The parenteral preparation may be enclosed in ampules, disposable syringes or vials made of single or multiple doses of glass, plastic or other suitable material.

If a compound shows insufficient solubility, methods for solubilizing the compound may be used. Such methods are known to those of skill in the art and include, but are not limited to,
Use of a co-solvent such as dimethylsulfoxide (DMSO), use of a surfactant such as Tween®, or dissolution in aqueous sodium bicarbonate. Also, derivatives of the compounds, such as prodrugs of the compounds, may be used in formulating an effective pharmaceutical composition. For ophthalmic indications, the compositions are formulated in an ophthalmically acceptable carrier. For ocular use herein, local administration, either by topical administration or by injection, is preferred. Also, a depressed formulation is desirable. Typically, the compositions are formulated for single dose administration so that an effective amount is administered in a single dose.

When the compound is mixed or added with the vehicle, the resulting mixture can be a solution, suspension, emulsion or other composition. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the chosen carrier or vehicle. If desired, pharmaceutically acceptable salts or other derivatives of the compounds may be prepared.

The compound is included in a pharmaceutically acceptable carrier in an amount sufficient to produce a therapeutically beneficial effect without undesirable side effects for the patient being treated. It is understood that the number and extent of side effects will depend on the condition to which the compound is administered. For example, certain toxic and undesirable side effects are tolerated in treating life-threatening diseases,
It would not be acceptable in treating less severe diseases. The concentration of the compound in the composition will depend on its adsorption, inactivation and elimination rates, dosage regimen, and dosage, as well as other factors known to those skilled in the art.

The compounds are also cardiovascular drugs for treating cardiovascular disease, stroke, infection, trauma and other diseases in which cell activation is involved in a causative or contributing role,
It may be mixed with other effective substances which do not impair the desired action, or substances which supplement the desired action, such as antibiotics, anticoagulants and other such agents known to those skilled in the art. Thus, advantageously, protease inhibitors such as futan also provide beta-adrenergic blockers (eg, atenolol), calcium channel blockers (eg, nifedipine), angiotensin converting enzyme (A
CE) inhibitors (eg, lisinopril), diuretics (eg, furosemide or hydrochlorothiazide), endothelin converting enzyme (ECE) inhibitors (eg, phosphoramidone), neutral endopeptidase (NEP) inhibitors, HMGCoA reductase inhibitors, nitric oxide donors In the treatment of one or more of the above-mentioned diseases or medical conditions, such as an antioxidant, a vasodilator, a dopamine agonist, a neuroprotective, a steroid, a beta agonist, an anticoagulant, or a thrombolytic. It may be administered together with other pharmacological agents known to those skilled in the art as valuable. It is understood that such combination therapy constitutes a further aspect of the compositions and methods of treatment provided herein.

Upon mixing and adding the compounds, the resulting mixture can be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the chosen carrier or vehicle.
An effective concentration is sufficient to ameliorate the symptoms of the disease, disorder or condition being treated, which may be determined empirically.

The formulations may be tablets, capsules, pills, powders, granules, sterile parenteral solutions or tablets containing a suitable amount of the compound or a pharmaceutically acceptable derivative thereof for administration to humans and animals. They are provided in unit dosage forms such as suspensions and oral solutions or suspensions, and oil / water emulsions. Typically, the pharmaceutically therapeutically effective compounds and derivatives thereof are formulated and administered in unit dosage or multiple dosage forms. Unit dosage form as used herein refers to physically discrete units suitable for human and animal subjects, which are individually encapsulated as is known in the art. Each unit dose contains a predetermined quantity of the therapeutically effective compound sufficient to produce the desired therapeutic effect, in combination with the required pharmaceutical carrier, vehicle or excipient. Examples of unit dosage forms include ampules and syringes and individually enclosed tablets or capsules. The unit dosage form can be administered in divided or divided doses. Multiple dose forms are a plurality of identical unit dose forms enclosed in a single container that can be administered in separate unit dose forms. Examples of multiple dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Thus, a multiple dose form is one in which there are multiple unit doses that have not been separated at the time of encapsulation.

The composition may contain, along with the active ingredient, an excipient such as lactose, sucrose, dicalcium phosphate or carboxymethyl cellulose; magnesium stearate;
Lubricants such as calcium stearate and talc; and natural gums such as starch, gum arabic, glucose, molasses, polyvinylpyrrolidine,
It may include binders such as cellulose and its derivatives, povidone, crospovidone, and other such binders known to those skilled in the art. Pharmaceutically administrable liquid compositions comprise, for example, an active compound as defined above and an optional pharmaceutical adjuvant, for example,
It may be prepared by dissolving, dispersing or otherwise mixing in a carrier such as water, saline, aqueous dextrose, glycerol, glycol, ethanol, and the like, thereby forming a solution or suspension. Also, if necessary, the administered pharmaceutical composition may contain trace amounts of wetting agents, emulsifying or solubilizing agents, non-toxic auxiliaries such as pH buffering agents, for example, acetates, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate , Sodium triethanolamine acetate, triethanolamine oleate, and other such agents. Methods of actually making such dosage forms are known, or will be apparent, to those skilled in this art, for example, as described in Remington's Pharmaceuticals.
ceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 19
See 75. The composition or formulation to be administered will in each case contain a fixed amount of the active compound in an amount sufficient to alleviate the condition of the patient being treated.

A dosage form or composition containing the active ingredient in the range of 0.005% to 100% with the balance consisting of a non-toxic carrier may be prepared. For oral administration, any of the commonly used excipients such as, for example, pharmaceutical grades of mannitol, lacrose, starch, magnesium stearate, talc, cellulose derivatives, croscarmellose sodium, glucose, sucrose, magnesium carbonate or saccharin sodium The mixture forms a non-toxic pharmaceutically acceptable composition.
Such compositions include solutions, suspensions, tablets, capsules, powders and sustained release formulations such as, but not limited to, implants and delivery systems encapsulated in microcapsules, and collagen, ethylene. Includes biodegradable, biocompatible polymers such as vinyl acetate, polyanhydride, polyglycolic acid, polyorthoester, polylactic acid and others. Methods for preparing these formulations are known to those skilled in the art.

The active compounds or pharmaceutically acceptable derivatives may be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation or coating.

Lastly, the compound provided herein or a suitable derivative thereof, and a compound or a suitable derivative thereof, which is effective to mitigate and reduce the activation of cells within the packaging material, the packaging material, can be used in a cell. A product such as a serine protease inhibitor such as futan can be encapsulated as a product containing a label indicating that the activity of the protein is reduced. The label may optionally include a treatment protocol that ensures a disease associated with cell activation or cell activation therapy.

H. Drug Screening Assays and Cell Activation Assays To screen for compounds that inhibit cell activation, pancreatic homogenates or fine fractions thereof, particularly those containing an active ingredient having a molecular weight of less than about 3 kD, are used. You may. The homogenate is contacted with suitable cells, such as an endothelial cell line or neutrophils, or a selected tissue, and the cells are assayed to assess activation levels. A test compound that reduces the level of activation can be identified by contacting the cell with the homogenate at the same time as, or after, contacting the cell with the test compound. Those that reduce the level of activation for the homogenate in the absence of compound are selected for further study. In another embodiment, the effect of a test compound is compared to known inhibitors, such as futanes and other serine protease inhibitors, with respect to the activity of the homogenate or a fraction thereof. Compounds that inhibit substantially or more potently than known inhibitors are selected for further evaluation.

Other Assays Donor cells or cell cultures, corresponding to a patient's blood plasma sample, were subjected to cell activation behavior, clastogenic (mutagenic) activity, apoptoti
c potential), which can be used to see the effects of intercellular junctions as related to the blood-brain barrier.

The isolation and identification of circulating plasma factors provides a specific assay with antibodies to these factors.

Another method is provided herein for assaying the neutrophils' ability to activate in a patient's serum as an indication of the presence of cellular activity (Reference Example 6). It can be used in a typical way. That is, the blood of a new patient is centrifuged and superoxide formation is measured. In other embodiments, control plasma of healthy individuals may be used as a vehicle to test various substances, including their plasma, for their activity. This latter method provides neutrophils to non-self plasma and eliminates the need for large patient plasma. The ability to activate other quiescent neutrophils can be measured on a small volume of 100 μl plasma (and can be reduced using a new small volume centrifuge). By this method, less than one hour
Accurate results can be obtained with (10 minutes centrifugation, 10 minutes setup and 40 minutes measurement). Since the number of neutrophils in spun plasma is much lower than the free neutrophils in autologous plasma, the relative levels of fluorescent luminescence are also reduced. Normal (control)
In plasma, all figures (up to 100 experiments with 5 or more donors) have been 20
Responses of up to 1500-6000 counts / sec were obtained within a -50 minute window. A typical range is about 3000 +/- 500 counts / sec for about 40 minutes.
This can be modified by the donor's condition, antibiotics and, more interestingly, the consumption of fatty foods.

These alone or in combination assays can be used to identify other factors and / or assess levels of cellular activity. They relate to the current state of the disease and can be used as support for effective treatment decisions. Other assays for measuring the level of cellular activity in a patient sample include any of the cellular activities known to those of skill in the art, and particularly those exemplified herein.

J. Generation of Additional Therapeutic Targets As described below, homogenates of tissues other than pancreas do not produce cell-active factors, but are treated by tissue treatment with pancreatic factors provided herein, and by treatment with proteases, particularly serine proteases. Activation occurred. Therefore, another target of drug screening is to treat the selected tissue with a pancreatic composition or an activated fraction thereof, or a protease inhibitor, and then use the pancreatic homogenate purification methods described herein to reduce the active fraction. It can occur by isolating and, ultimately, obtaining active factors from other tissues.

Assay System for Measuring Cell Activity Summary Methods for measuring plasma cell activity in vitro have been developed. It has been applied to 96-well plates and forms suitable for automated spectrophotometric analysis with commercially available ELISA readers (eg, Pick et al., (1981) J. Immunol. Methods. 46
: 211-226).

Description As described herein, hydrogen peroxide is a common reactive oxygen species (ROS) found in patients and animals under oxidative stress. This product is a relatively stable end product of the free radical oxidation of the superoxide anion (O ₂ ⁻ ), and as such is an important indicator of disease progression and other etiologies. The test provided herein is used for the spectrophotometric measurement of a substrate chromogen whose color changes upon oxidation by hydrogen peroxide. The resulting color change is measured by the visible spectrum up to 610 nm. Several measurements can be performed to evaluate various parameters indicative of various diseases or conditions.

The blood collected from the patient at risk is centrifuged at 500 g and the plasma layer contained in the buffy coat containing the leukocyte-rich band is collected with a sterile pipette. This plasma is
Pipette into wells of 6 wells or higher density plates at a volume of about 100-200 microliters. In addition, all blood samples can be added directly to reagents in a 96-well (or higher density) plate. The mixture was centrifuged and an aliquot of (reacted) plasma was collected and measured.

This method is modified for automation. The most effective measurement scheme involves three measurements on the same sample, each with a separate mediator. If necessary, one measurement per patient is sufficient and provides satisfactory clinical information.

[0146] Plasma is added to the wells. Each contains chromogens and oxidants. For example, phenol red (sodium salt) is selected as the substance chromogen and the enzyme horseradish peroxidase is selected as the oxidizing agent. Therefore, such other substances and enzymes are replaced (Pick et al., (1980) J. Immunol. Metho
ds 38: 161-170; Pick (1981) J. Immunol. Methods 46: 211-226). The three measurements per patient can be distinguished as follows:

(A) The first measurement sample contains only the above substances. This provides a baseline activation level when compared to other samples. This sample is then evaluated against a control sample (third measurement).

(B) The second measurement sample contains (in addition to the above reagents) superoxide dismutase (SOD), an enzyme specific to superoxide. Therefore, the dismutation reaction proceeds, producing more hydrogen peroxide, and the difference between this value and the second measurement sample is the amount of ROS produced by circulating cells and potent circulating enzymes.

(C) The third measurement includes (in addition to the above reagents) enzymes specific to catalase and hydrogen peroxide. This sample serves as a control sample for the measurement and therefore does not contain any hydrogen peroxide. Add sodium hydroxide (1N) to all samples,
Adjust the pH.

By using three sample measurements per plasma sample (approximately 300 microliters in volume), many treatments can be determined for a patient's cardiovascular health. An increase in the level of the first measurement as compared to a control sample (third measurement) indicates the presence of an inflammatory disease (activation).

A second measurement determines the location of this inflammation. Elevated first and second sample measurements indicate up-regulation of leukocytes, which usually corresponds to the onset of disease or the onset of acute cardiovascular disease such as ischemic complications. On the other hand, a high first measurement and a low second measurement indicate the presence of a chronic or immune compromised condition such as hypertension or sepsis.

The typical advantages of this measurement are that fewer samples are needed for the test, the standard EL
The ease with which the experimental system can be performed using ISA, and most importantly, the ability to obtain relatively accurate information about the status of cardiovascular activation affecting an individual.

The following examples are for illustrative purposes only and do not limit the scope of the invention.

Example 1 Introduction This example shows a brief introduction of a study directed at neutrophil activators.

As described above, factors that induce or cause a pathological condition in a living body are created in the body. Disease causes upregulation of host defense responses by cells in the body (circulating cells and tissues (eg, endothelial cells, mast cells)). Prominent among the cells that are up-regulated are leukocyte neutrophils, which potentiate the body itself in its ability as a second line of defense (after the border of the body's skin and oral mucosa). Has the ability to hurt. There is just a good line between the easy destruction of the invading pathogens and the phagocytosis, which is necessary for the survival of the organism and is unsuitable for the activation of these cells. In the past, before the arrival of antibiotics, deaths from sepsis were very common. The more activated the neutrophil response to invading bacteria, the more chance the host had to survive the attack. Because antibiotics have greatly reduced mortality in the response to infection, people are prone to longevity and are susceptible to diseases such as atherosclerosis and hypertension, which are rare in young people. Thus, there is a trade-off between neutrophil activation required for prevention of external infection and inflammatory regulation important to avoid fundamental damage to the host. While defense against pathogens is needed, neutrophil activation may also respond to tissue destruction and organ damage seen as a result of its activation. This “autoimmune response” includes many medical conditions, including cardiovascular disease (myocardial infarction, stroke, atherosclerosis, hypertension), arthritis, sepsis, traumatic shock, and shock . The causes of up-regulation of cells in the body, seen in response to various stimuli, are not well understood. Indeed, upregulation of host defense involved many mediators, and activation of neutrophils by innumerable factors, cytokines and bacterial products was observed in vitro and in vivo, but became normal or systemic No mediator is clearly identified.

Neutrophil activation not only serves as a host response to foreign antigens, but also includes reactions that are often detrimental to the host. Studies have focused on factors that activate neutrophils in vitro and in vivo (Wientjes
et al., (1995) Semin Cell Biol 6: 357-651; Ley (1996) Cardiovasc Res 32:
733-42; Downey et al., (1995) Semin Cell Biol 6: 345-356). Most of the research in this publication has described the triggering mechanisms that upregulate these cells as
Assumes a sudden event or recurrent chronic disease. Activation of neutrophils
Occurs in the absence of recognizable pathogens, and the resulting "pre-activation" can have harmful effects on the host during trauma or other stressor events. Preneutrophil activation appears to be seasonal in nature, with peak activity levels in winter,
The minimum is summer. This may be related to the observed seasonal increase in circulating factors that can be harmful to the body and mind, including lipids and fibrinogen. In addition, daily changes, exercise, and especially diet can affect baseline levels of neutrophil activation and can affect circulating levels of (neutrophil) inflammatory products such as superoxide. For example, plasma from an otherwise healthy blood donor who ate a diet rich in saturated fats the night before was upregulated compared to plasma from the same subject on a low fat diet. Neutrophil activation was observed.

The mechanism that affects neutrophil quiescence is due to a combination of factors, so that neutrophils change very sensitively in the surrounding environment and are readily active.

1.1 Pre-Activation and Priming The knowledge that activated neutrophils are involved in the pathogenesis of the disease has led to the link between neutrophil activation and acute trauma, as injury to hemorrhagic shock or acute endotoxemia. And was investigated herein. Neutrophils are not only activated systemically by shock, but also plasma factors whose activation is induced upon incubation with natural donor neutrophils,
This proves the critical presence of circulating neutrophil activators (Barroso
-Aranda et al. (1992) Circ Shock 36: 185-190; Barroso-Aranda et al. (198
9) Am J Physiol: H846-852; and Shen et al. (1990) Circulatory Shock 31
: 343-344).

[0159] Neutrophils circulate in vivo as a heterogeneous group that includes non-activated, "primed" and activated cells. A primed cell is a cell intended for a subthreshold stimulus and is hyperresponsive to any additional stimulus. Some researchers maintain neutrophils to require priming before they can be activated in vitro (ie, the necessity of two stimuli), and there is evidence to support this. On the other hand, any stimulus of sufficient magnitude also directly stimulates neutrophils. Therefore, the relative importance of in vivo priming has not yet been elucidated, and it is not known how circulating neutrophil activators may be “primers” to boosters. Most likely, the circulating neutrophil activator shifts the population distribution for more activated and primed cells at the expense of the non-activated population. This is an experiment that measures neutrophil superoxide production (an indicator of neutrophil activity) in animals with hemorrhagic shock (Barro
so-Aranda et al., (1989) Am J Physiol: H846-852). Superoxide levels are increased before and after shock in non-surviving animals compared to surviving animals, which results in a large number of primed and activated cells before injury (shock) with higher levels of activated cells after injury. (Because all activated cells are a combination of primed and activated neutrophils).

However, a more interesting finding is that early levels of neutrophil activation by circulating plasma factors are inversely correlated with the survival of shocked animals compared to other identical animals. . This is because in other suitable subjects there is a difference in the level of neutrophil activation in individuals that can result in high levels of mortality in many neutrophil "pre" activation subjects. We propose that activation is due to some of the circulating humoral factors.

It is proposed herein that this activation is central to the interest of therapeutic intervention and also in treatment protocol evaluation.

1.2 Purpose Under well-controlled experimental conditions, significant differences in tissue damage and viability after shock with other suitable subjects occur. In animals with high initial levels of preactivator, not only hemorrhagic shock,
Septic shock also reduces survival. These preactivators appear to stimulate (up-regulate) cells of the cardiovascular system. The biochemical properties of the factor have not been fully elucidated, but appear to be substances transported in plasma. Although the response is small with low levels of activators, plasma with high levels of preactivation activates untreated neutrophils. Therefore, it is determined by superoxide production and actin polymerization tests.

The present invention suggests that these activators increase oxygen free radical production during reperfusion, in addition to increasing shock mortality, resulting in high levels of lipid peroxidation and cell death. Cause. These activators are endogenous in tissues and are released in sub-clinical perturbation to tissues, most notably in response to food, exercise, stress, and exogenous pathogens. Circulating activators shift the distribution of neutrophil populations to the activated state, increase tissue damage in chronic conditions and increase acute symptom modality. Thus, once that level is determined, the treatment modality and the outcome of the treatment are based on the assessment of these levels.

1.3 Significance of Endogenous Neutrophil Activators The importance of neutrophil activators produced during shock and found endogenously in tissues is described herein. An understanding of the function of these factors in vitro and in vivo provides a better understanding of their effects during shock and in healthy individuals. With an understanding of their mechanism of action, strategies have been invented to prevent inappropriate neutrophil activation by these factors, either in the form of acute interventions or in the form of daily regulation in the maintenance of health care.

Example 2 Activation of Neutrophil Cells: Determination and Quantification Neutrophils are involved in many disease processes, acute and chronic pathologies. For these cells to have detrimental effects on the host's mind and body, they must first be activated. "Activation" of neutrophils can be quiescent or "involved" when it involves up-regulation of oxidative metabolism, increased intracellular calcium concentration, morphological changes induced by cytoplasmic protein polymerization, and ultimately the breakdown of cytoplasmic particles. Normally changes state. These processes in vivo are not linked, and various stimuli can trigger varying degrees of up-regulation of these parameters.

Thus, the term “activation” must clarify the term for a particular parameter. In these studies, superoxide production (as measured by the nitroblue tetrazolium test and lucigenin-stimulated chemiluminescence) and actin polymerization (as measured by the pseudopodia formation test) are indicators of neutrophil activation. chosen. These two responses are separate. In dormant neutrophils, there is a slight correlation between "activation" as measured by the two types of measurements. This correlation clearly increases due to increased stimulation of neutrophils. Thus, the use of two parameters in the definition of "activation" will broadly evaluate neutrophil up-regulation.

2.1 Method of Assessing Neutrophil Cell Activation Neutrophils “activate” when exposed to a solubilizing stimulus. Neutrophil activation involves actin polymerization, superoxide polymerization, cytolysis and protease release (Ferramt
e et al., (1992) Immunol Ser 57: 499-521; Ley (1996) Cardiovasc Res 32:
733-42, Chatham et al., (1994) J. Leukoc Biol 56: 654-660), and upregulation of adhesion molecules (Ley (1995) Bioeng Sci News 18: 43-47; Muroh
ara et al., (1995) Cardiovasc Res 30: 965-974; and Jaboson et al., (199
3) Represented by a number of parameters that are up-regulated under the symptoms of inflammation, including J. Immunol. 151: 5639-5652). Although these indicators of activation need not be connected, neutrophils tend to exhibit all of these characteristics when targeted at important stimuli.

In the case of these studies, actin polymerization, superoxide and hydrogen peroxide formation were used to determine the activation status of the neutrophil population. This response to stimuli can form in various ways, including oxidative burst mechanisms (NADPH oxidase), actin polymerization (from spherical or g-actin to fibrous or f-actin), expression of adhesion molecules and lysosomes. Includes particle disintegration. The mechanism is up-regulated in part in response to stimuli to which neutrophils are exposed. For example, leukotriene B ₄ (LTB ₄ )
And the complementary fragment C5a are potent chemoattractants, but are poor stimulators in the acidification response, such as ATP.

Other cytokines such as interleukin-1 (Il-1), neutrophil-activated protein-1 / interleukin-8 (NAP-1 / Il-8), tumor necrosis factor-α (T
NF-α), granular leukocyte / macrophage colony stimulating factor (GM-CSF), and gamma interferon (γ-IFN) are also poor direct stimulators of NADPH oxidase. However, these factors, such as the bacterial product lipopolysaccharide (LPS), are potent "priming" agents and enhance the acidification response to other stimuli.

Environmental factors such as permeability changes and excessive shear stress also prime neutrophils, but conversely, physiological levels of liquid shear need not cause neutrophils to be up-regulated in circulation. It turned out to be. The normal activating peptides formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet activating factor (PAF) do not activate the respiratory burst of isolated neutrophils in the absence of plasma.
(See Example 4), suggesting the need for sufficient intracellular (PMN) ATP to activate respiratory bursts in response to these stimuli.

In most inflammations, there is simultaneous, but not all, up-regulation of neutrophils by most, if not all, stimuli by either multiple stimuli or excess one stimulus (Badwey). et al., (1991) Adv Exp Med Biol 31
4: 19-33). There is not much correlation between tests on non-activated neutrophils. There appears to be a minimal correlation between actin polymerization (pseudopodia formation) and acidification burst as measured by the NBT test on quiescent neutrophils in fresh whole blood samples.

The test adds the plasma of rats suffering from SAO shock to isolate human neutrophils for pseudopodia formation tests, and adds the same plasma to whole rat (donor) blood for NBT tests This was implemented. A similar correlation is seen in a repeat test using whole human blood for the NBT test.

For the studies described herein, neutrophil activation is defined by acidification burst and actin polymerization. Acidification burst components were measured using lucigenin-enhanced chemiluminescence and the nitroblue tetrazolium (NBT) test. All of these tests are sensitive to the production of superoxide and can be inhibited by superoxide dismutase (SOD). The test for actin polymerization is based on the detection of pseudopodia formation, with cell transformation from spherical to polarized form.

With the combination of these two assays, the two components of neutrophil activity can be measured, and usually correlate when assessing cells exposed to a stimulus. Both types of tests have strict sensitivity, especially NBT and pseudopodia formation tests are limited to less than two orders of magnitude. Measured values range from 0 to 100% because the total cell count is 100 cells for each test. However, the use of two assays results in "activation" and "
The classification of the "non-activated" cell population is appropriately assured.

2.2 Acidification Bursts Neutrophil oxidative bursts are due to the membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, turning oxygen into superoxide (free radicals) NADPH + 2O ₂ → NADP ⁺ + 2O. ₂ ^{- +} H + ₍₂₎ reaction is converted through the. Free radicals and unpaired electron-associated molecules are reactive in nature and are known to cause tissue damage resulting in cell membrane breakage, protein denaturation and nucleic acid degradation (see also Example 3.1.b). ). Superoxide and its dismutation product, hydrogen peroxide (H ₂ O ₂ ), is the oxygen free radical component formed by activated neutrophils. Superoxide is essentially non-reactive, although it is thought to be produced predominantly extracellularly, but does not readily cross cell membranes except for the assumption that it is through an ion channel. Hydrogen peroxide, on the other hand, is more stable and is able to pass freely across cell membranes, but is minimally toxic at physiological concentrations (<1 mM) and reduces the degree of acidified cell damage suffered by activated neutrophils. Unexplainable (apart from granule loss). The interaction between these two species is thought to produce the cytotoxicity of free radical-induced oxidation, catalyzed by iron and other divalent metals that effectively react with all biological materials to form strong hydroxyl radicals. is there. O ₂ ^- is produced in large amounts and 2 × 10 ⁶ neutrophils stimulated with 10 ⁻⁸ M fMLP
In a volume 1-2μl, 1 minute, O ₂ of 10 nmol ^- has been reported to produce. This is about 5-10 mM O ₂ ^- corresponds to the product of / min. Superoxide spontaneously dismutates to hydrogen peroxide (even at low rates) or even faster in the presence of superoxide dismutase (SOD).

The superoxide and hydrogen peroxide are then reacted in a Haber-Weiss reaction, producing highly reactive hydroxyl radicals. O ₂ ⁻ + Fe ³⁺ → O ₂ + Fe ²⁺ Fe ²⁺ + H ₂ O ₂ → Fe ³⁺ + OH ⁻ + OH ⁻

However, the Haber-Weiss reaction occurs slowly in vivo (Halliwell et al.
l., (1990) Methods Enyzmol. 186: 1-85), catalyzed by transition state metal ions. In metal-catalyzed Haber-Weiss or Fenton reactions, it is believed that superoxide and hydrogen peroxide contribute to cell death.

[0178] This theory, O ₂ ^- SOD by inhibiting and hydrogen peroxide formation, respectively
And the observation that catalase, an enzyme that breaks down hydrogen peroxide to O ₂ + H ₂ O, reduces neutrophil-mediated acidification damage in many systems. The inhibition is
Either using only SOD, using only catalase, or using a combination of the two is usually more effective. Depending on the organ and injury model studies, differences were made with respect to the relative effects of each inhibition. The ultimate toxic component of the neutrophil respiratory burst is hypochlorous acid (HOCl) formation, which is caused by the reaction of a halogen such as chlorine with H ₂ O _{2 in} the presence of the neutrophil azulogenic granulase myeloperoxidase. It is formed.

2.3 Method: Measurement of Acidification Burst Quantification of neutrophil acidification burst is performed by reaction of superoxide with other substances, either lucigenin or nitroblue tetrazolium, a product that can be easily measured. Generate NADPH oxidase is activated via a number of mechanisms, receptor-mediated and non-receptor-mediated. Examples of receptor-mediated stimuli include fMLP, C5a and TNF-α. Non-receptor-mediated stimulants include calcium ionophores, protein kinase-C (PKC) activators such as phorbol myristate acetic acid (PMA), G-protein agonists and surfactants such as surfactants and arachidonic acid. Contains irritants.

In the experiments of NADPH oxidase-mediated cell activity described herein, the known receptor-mediated activators PAF and fMLP and pancreatic activator compositions provided herein were used.

2.3. a Chemiluminescence Assay A chemiluminescence assay using lucigenin to promote superoxide producing chemiluminescence is discussed in detail in Example 6.

2.3. b Nitroblue tetrazolium test In the NBT test, the counts create the percentage of circulating neutrophils of untreated donors that can naturally reduce blue-yellow NBT to blue-black formazan crystals in the presence of donor plasma (Shen et al. al., (1990) Circulatory Shock 31: 343-344). NBT reduction by neutrophils is accompanied by enhanced superoxide formation (Shen et al., (1990) Cir.
culatory Shock 31: 343-344). Pretreatment with superoxide dismutase (bovine erythrocytes, Sigma Chemical Company, St. Louis, MO) inhibits this reaction
(Barroso-Aranda et al., (1989) Am J Physiol: H846-852).

To perform this test, 0.4 ml aliquots of test plasma or activator are collected for each sample. Fresh arterial blood (0.1 ml) of donor animals or heparinized human venous blood of healthy volunteers is mixed with 0.4 ml of test plasma or activator and immediately cleaned in a clean siliconized 1-dram glass vial (Sigma Diagnostics, St.
(Louis, MO) and mix with an equivalent 0.1% NBT-solution. This mixing method avoids the step of spontaneously inducing activation, the centrifugation of donor neutrophils. About 40
A percentage of 0.1 ml of whole blood and 0.4 ml of plasma with a% hematocrit would expose the donor neutrophils to a concentration equivalent to at least 80% of the plasma of the test (eg, shocked) animal. Plasmapheresis is not accompanied by abnormally visible red blood cell reactions or cell aggregation. The glass vial is then incubated in air at 37 ° C. for 10 minutes, then left at room temperature for another 10 minutes. At the end of this time, the blood-NBT mixture is gently agitated. A cover glass smear is prepared and stained with Light's stain. 1000 neutrophils in total 1000 x oil objecti
Count as usual with ve magnification. Neutrophils with spotted cytoplasm with formazan coating or formazan density aggregation are counted as NBT positive cells, slides are measured twice and three times, and the results are averaged. Light micrographs of typical non-irritating rat neutrophils in non-activated rat donor plasma show no NBT crystals. Cells are stained with Wright's stain. Light micrographs of rat neutrophils stimulated by the addition of activated rat donor plasma show NBT crystals in the cytoplasm.

In a modification of the standard NBT test protocol, the crystal violet nuclear stain is replaced with Wright's stain. This staining has advantages only in the case of nuclear staining of white blood cells, making neutrophil identification a relatively simple process. Care must be taken in the use of this stain. If not applied carefully, it tends to solubilize in the blood smear. This staining and the use of NBT (formazan) in a standard "wet volume" (isolating PMN for pseudopodia determination) allows for the accurate visualization of NBT crystals not only in pseudopodia but also in untreated spherical cells. Caused by Wright
Shows that the percentage of NBT (+) cells is higher than normal detection using t's staining. In the case of NBT (+) using the latter method, almost 100% of quiescent cells
Becomes This confirms that non-activated neutrophils continuously produce at least basal levels of superoxide. NBT counts using crystal violet staining mean where applicable, and care must be taken to directly compare these counts with previous results using Wright's staining.

2.3. c Peroxide production measurement In some plasma samples, the concentration of peroxide, probably attributable to hydrogen peroxide, was measured. Although there is a (circulating) product of peroxide in the isolated plasma (see, eg, Example 3), it is believed that many of this radical product comes from superoxide producing neutrophils. Using electrode technology, the level of peroxide (hydrogen) produced in activated plasma after catalase inhibition by sodium azide can be measured. Briefly, this method uses a platinum working electrode and a platinum counter electrode for a silver / silver chloride reference negative electrode. The difference in voltage was maintained at 0.6 V between the reference and working electrodes while a current was flowing between the reference and working electrodes. Hydrogen peroxide in the sample
When reacting on the surface of the working electrode, H ₂ O ₂ produces O ₂ , 2H ⁺ and 2e ^−, thus producing a current.

A sample of heparinized blood (1.5 ml) was removed, divided into two 0.75 ml aliquots and placed directly on ice (0-4 ° C.). They were then incubated for 10 minutes at 37 ° C. and centrifuged for 10 minutes at 500 G at room temperature. Measurements were then made in the supernatant plasma fraction with sodium azide (20 μl of a 2M stock solution) added to the first sample plasma layer and with the catalase (enzyme (3.2M (NH ₄ ) Aspergillus niger in ₂ SO ₄ , pH 6.0, Sigma C
chemicals, St. Louis, MO) in 20 μl containing 0.25 mg). After gentle stirring, the electrodes are placed in an appropriate aliquot of plasma and the output is recorded for 10 minutes, at which time the signal is at a steady state. Sodium azide (Sigma Chemical Co.
mpany, St. Louis, MO) inactivates plasma enzymes, including catalase, and degrades hydrogen peroxide. Catalase degrades hydrogen peroxide to oxygen and water. for that reason,
The current obtained from the sample with catalase is subtracted from the sample with sodium azide, and the difference in current results in hydrogen peroxide production.

Exogenous hydrogen peroxide in blood samples is measured with an electrode chemical sensor. The measurement is
Performed in supernatant plasma with sodium azide and catalase. Subtracting the current measured in the catalase sample from the current in the azide sample produces a current resulting from the hydrogen peroxide in the sample. The sensor was 0.6 for the silver / silver chloride negative electrode.
It has a platinum positive electrode that provides a V bias. Hydrogen peroxide reacts on the surface of the positive electrode and produces an electric current in proportion to the peroxide in the solution.

The system is calibrated with two plasma samples (containing 20 mM sodium azide) with the electrodes placed in 2 ml buffered saline solution. A known concentration of hydrogen peroxide is added to the solution and the electrode current is monitored. 0 linear response to current
It occurred at 10 μM. Equation to determine the actual peroxide concentration is to measure the least squares fit from the calibration curve (least-square fit), determined in peroxide concentration (μM) = (V _azide -V _{Katarase Bu)} ^* 10.43 + 0.45 I do.

2.3. d Pseudopodia formation Neutrophils (PMN) with pseudopodia due to actin polymerization using pseudopodia formation measurement
Measure the percentage of The difficulties described below arise when it is determined that pseudopodia formation can occur due to non-specific cell membrane activators such as detergents. Care should be taken and the activator removed.

To isolate human neutrophils, plasma is separated from erythrocytes by sedimentation and neutrophils are isolated by the Percoll-gradient technique. Because neutrophils are sensitive to changes in the physical environment, they must be especially careful not to vigorously stir cells.
Care includes conventional dextran-70 sedimentation techniques and changes in the permeability of buffers to lyse red blood cells. In these techniques, the neutrophil layer is clearly not activated, but is activated by this type of treatment.

For the pseudopodia assay, venous blood is collected from healthy volunteers in heparinized tubes and kept on ice. It is important to use heparin as an anticoagulant and not EDTA. This is because the calcium chelation property of EDTA affects neutrophil activity. Rat neutrophils are denser than rat erythrocytes, which makes isolation of neutrophils of rat PMN a considerable waste and difficult. After sedimentation in an appropriately sized sterile syringe, 3.5 ml of the minimum erythrocyte, including leukocytes, is added to the plasma while cooling on ice (60 ml).
Layered in Histopaque (Sigma Chemical Company, St. Louis, MO) and placed in a 12 ml polypropylene centrifuge tube (17 x 100 mm, Falcon, Shrewsbury, MO).
Layer the liquid in MA) and centrifuge at 600 G for 20 minutes. The sedimented erythrocytes and neutrophils were washed with 2 ml D-PBS or other Krebs-Henseleit buffer (11
8 mM sodium chloride, 4.7 mM potassium chloride, Tristrizma-HCl (Sigm
a Chemical Company) to a pH of 7.4). Other chemicals of Krebs-Henseleit buffer are available from Fisher Scientific, Fair Lawn, NJ. The resuspended cells were then added to 55% isotonic solution Percoll (Sigmal Ch.
emical Company) and slowly layer on 2.5 ml of 74% isotonic solution Percoll in deionized water. The suspension was centrifuged at 15 min 600G, as the resuspended in granulocytes layer was removed in PBS medium, a concentration 10 ⁶ neutrophils / ml. Aliquot 100 μl of suspended neutrophils into 100 μl of test plasma or active agent
Add to l. The mixture is mixed and then incubated for 10 minutes at 27 ° C. After incubation, 100 μl of 3% glutaraldehyde (Fisher Sc
ientific) to stop the reaction. Then, 100 μl of crystal violet in a phosphate buffer (pH 7.4) was added, and wet mount preparation (wet mount) was added.
t preparation) to stain the nuclei of leukocytes. Neutrophils with pseudopodia are suspended in large quantities and identified by the presence of segmented nuclei and cytoplasmic granules. Count 100 neutrophils. Cells with pseudopod-like protrusions of about 1 μm or more are considered positive. Repeated measurements indicate that the count reproducibility is within 2%.

For some experiments using the pseudopodia formation test, the following modification procedure was used. The results from the two methods are slightly different, most likely due to differences between observers
(Table 2.1 below).

2.3e Neutrophil Isolation in Ficoll / hypaque Two media (A and B) can be used, a single medium (A) or a discontinuous gradient. In the case of medium A, Ficoll 400 (Pharmacia no. 17-0400-0)
1, Piscataway, NJ) is dissolved in 440 ml of water (this yields 460 ml of solution). The density of the solution is determined with a pycnometer (about 1.0303 g / ml at 20 ° C.) and then sterilized. High Park-76 (Sanofi / Winthrop no. NDC 0024-007
24 ml of 76-04, 66% meglumine diatrizoate and 10% sodium diatrizoate, 1.432 g / ml) are added to 100 ml of this solution. Remove 15 ml of the mixture and measure the density again with a pycnometer. The value obtained is 1.10
61-1.1063 g / ml. The density can be reduced or increased and adjusted, respectively, by adding more Ficoll solution or Hyperc. This medium is slightly hypertonic. Medium B is Ficoll-Paque (Pharmacia no. 17-0840-03, Piscataway, NJ) which is commercially available for lymphocyte isolation and is less dense than medium A. It contains 5.7 g of Ficoll 400 and 9.0 g of sodium diatrizoate per 100 ml of solution.

Table 1 Table 2.1 Comparison of different pseudopod methods A comparison of the pseudopodia formation assay results from the two methods described in this document obtained by various observers. These assays were performed on HPLC separation columns. Large differences in a single sample ( ^* ) gave different interpretations of surfactant-induced (TFA and acetonitrile) polarization.

The whole blood is drawn from a (human) ^* donor into a syringe containing EDTA pH 7.3 (final concentration 10 mM). In a 50 ml conical tube, 30 ml of this blood was added to medium B2.
Layer with -3 ml and 12 ml of Medium A. The tube is then spun at 750 G for 25 minutes at 20-24 ° C. at the end of 25 minutes to prevent disruption of the layer without breaking the centrifuge spinning head. Neutrophil bands (mononuclear and erythrocyte) are removed and washed with Earl Balanced saline solution (EBSS) without calcium or magnesium and containing 9 mM morpholinopropanesulfonic acid (MOPS) pH 7.35. The second wash is performed with a 1: 1 mixture of EBSS without Ca ²⁺ and Mg ²⁺ and normal EBSS (both with MOPS). Cells
Ultimately incorporated into normal EBSS, including MOPS, pseudopodia formation is counted and investigated. By this method, about 6-30 × 10 ⁶ neutrophils are obtained per 10 ml of whole blood. Contaminating cells are mainly erythrocytes with some mononuclear cells (1-5 of isolated leukocytes).
%). The entire isolation procedure takes 90 minutes.

Count neutrophils, dilute to 1.1 × 10 ⁶ / ml and leave at room temperature for 5 minutes.
100 μl of activator (pancreatic homogenate, fMLP, etc.) is added to 900 μl of suspension at a concentration set to the final treatment concentration. Start the timer,
After 2 minutes, 100 μl of this suspension is added to 125 μl of ice-cold glutaraldehyde (2.5% in 0.9% NaCl) in the wells of a microtiter plate. The cells are allowed to cool, sediment, count (100 per well), and measure the percentage of polarized neutrophils. Cells are examined under 400x and those that deviate from the typical sphere are evaluated as polarized. Results are expressed as the percentage of polarized cells per total cells counted (100 cells per sample, except where indicated).

Most cell separation methods were performed using original separation techniques. Variations on this method are indicated when applicable.

2.4 Discussion The practice of the diagnostic and treatment evaluation methods provided herein requires that the activity of neutrophils to various stimuli in vitro and in vivo be accurately determined. The in vitro techniques discussed herein represent some of the currently used methods for assessing neutrophil activity. Other methods may also be used.

Problems arise when they rely on a type of test result, since they are biological assays and the NBT and pseudopodia tests depend in particular on the objectivity and accuracy of the observer. As such, one or more tests are often used, especially when the results are not clear. Also, as discussed above, the various formations of neutrophil activation in response to a stimulus are not necessarily combined or occur normally. Thus, in the case of the experiments described herein, within the practical constraints of time and substance, four tests are applied as much as possible to more accurately measure the magnitude and nature of neutrophil activation.

As noted above, neutrophils are very environmentally sensitive and are easily activated. Activation, as assessed by NBT and pseudopodia formation tests, also requires duality. That is, the cells are either activated or not activated.
Under normal environmental conditions, this does not occur, but problems arise when exposing the cells to a non-physiological environment, such as high performance liquid chromatography (HPLC) that filters the sample or separates the organic / inorganic phase. This is the case where the activation was measured using.

Therefore, care must be taken to ensure that the medium in which the activation measurement is performed is as physiological as possible. This typically involves a dose-dependent test, in which the amount of acceptable solvent is determined during a given measurement episode. If not suitable for discussion, these calibration tests will not be reported here. However, the activation of a control sample with a calibrated amount of solvent, itself, is reported and classified.

Example 3 Presence of Hemorrhagic Shock and Activators Summary Hemorrhagic shock involves the coordinated action of activated neutrophils, oxygen free radicals, inflammatory cytokines and other circulating mediators, lipid peroxidation and cell death. A well-studied model of acute trauma involving the resulting unregulated product. In this global ischemic reperfusion typical, upregulation of activators, lipid peroxidation and cell death in shock plasma, measured as an increase in plasma peroxide levels, not only occur during the reperfusion component, but also to some extent It was also observed during periods of hypotension. The association of these groups is not only synergistic between their actions, but also raises questions about the usual assumptions about the transient progression of hemorrhagic shock. In particular, the inclusion of activators during the shock process and in "pre-activation" of plasma before shock has proven to be a major determinant of the process and progression of acute trauma.

3.1 Introduction 3.1a Hemorrhagic Shock Global ischemia and secondary reperfusion result in complications accompanied by cell and organ damage. Tissue damage after ischemic shock can include the degree of pressure reduction, anesthesia (if possible)
And the duration of ischemia and the nature of the affected organ. Some organs, prominent skeletal muscle, survive up to 4 hours of ischemia without adverse effects. Others, such as visceral areas and the brain, are more sensitive and cannot tolerate long periods of low flow conditions.
Organs such as the heart cannot tolerate limited ischemia for a short time.

[0203] There is a clinically relevant time window where reperfusion is desirable. Not all cells die, but rescue is possible. Interventions for ischemia reperfusion, including hemorrhagic shock, must occur during the "treatment window" or before rescuing the tissue. After this, the injury is irreversible with respect to the intervention (Sus
sman et al., (1990) Methods Enzymol 186: 711-783). Shorter reperfusion after ischemia results in less loss of tissue function, while longer ischemia
Causes cell death and tissue necrosis. In that case, reperfusion or not. Contrary to low flow conditions, total occlusion of blood vessels results in predominantly anoxic cell death other than free radical interactions when reperfusion is not available (McCord (1986) Adv. Free Rad Bio &
Med 2: 325-345).

In normal hemorrhagic shock and ischemic conditions, reduced blood flow results in reduced oxygen transport to tissues and reduced excretion. These factors weaken the function,
Eventually, the organization dies. Paradoxically, the replacement of blood in the case of hemorrhagic shock, or the restoration of blood flow to pre-ischemic tissue, results in a phenomenon known as "reperfusion wound". This wound results from prior re-oxygenation of ischemic tissue and the production of oxygen free radicals and other toxic substances. Free radicals, molecules with unpaired electrons, are highly reactive and are known to cause tissue damage resulting from cell membrane damage, protein denaturation and nucleic acid destruction. Although oxygen free radicals affect reperfusion wounds, the free radicals are included and the location of the product is not determined. According to a strong hypothesis, hypoxia due to low blood flow and subsequent oxygen exchange in ischemic tissue results in the activation and upregulation of other good enzymes and the production of large amounts of free radical species.

3.1. b Oxygen Free Radical Generation The major component of these free radicals is thought to be the endothelium-bound enzyme xanthine oxidase (XO), and is subjected to limited proteolysis to react with xanthine dehydrogenase (XD) using NADH. It is converted either reversibly or irreversibly to xanthine oxidase using O ₂ . XD due to hypoxia
It is converted to O, whereas increased ATP metabolism increases both XO / XD substances, xanthine and hypoxanthine. In reperfusion, oxygen is again readily available again, and xanthine and hypoxanthine are destroyed by XO, producing uric acid. In the process, free radical superoxide (O ₂ ⁻ ) and hydrogen peroxide (H ₂ O ₂ ) are released in a ratio of about 30:70. By reaction of xanthine (and Hipokisa Nchin) and xanthine oxidase, superoxide and hydrogen peroxide occurs: xanthine _{+ 3O 2 + 2H 2 O 2} → 2O 2 - + 2H + + H 2 O 2.
In the quiescent state, xanthine oxidase exists as xanthine dehydrogenase and reacts with NAD + to form NADH and uric acid.

Circulating XO is also involved as a component of global ischemia / reperfusion wounds. Other sources of oxygen free radicals include mitochondrial cytochromes, which are probably inactivated by ischemia and NADH oxidase. Included among reperfusion oxygen free radical reaction, a neutrophil, O ₂ through the membrane bound NADPH ^- secrete similarly emit host membrane disruption proteases and other substances (see Example 2) . With respect to its mechanism, superoxide and hydrogen peroxide appear to be the major oxygen free radical components formed by reperfusion wounds.

However, the question regarding “toxic radical free radical theory” stems from the differences in the tissues and species studied and experimental protocols. For example, rats appear to have high levels of XO in tissues such as the intestine, which reveals a wound on intestinal ischemia / reperfusion. In contrast, XO has been reported to produce only small amounts in human heart tissue. Despite this evidence, the XO inhibitor allopurinol is effective in mitochondrial ischemia / reperfusion, apparently attributable to other effects of the drug or potent inhibition of circulating XO. There is the question of why SOD reduces tissue wounding when the relative ratio of hydrogen peroxide, which is more toxic than superoxide, actually increases. This is probably explained by proper catalase levels in certain tissues, which can inactivate these increased levels of hydrogen peroxide. Because superoxide is required for the Fenton reaction, application of SOD mitigates oxygen wounds by removing one of the necessary components of this reaction.

Lipid peroxidation is “oxidative degradation of polyunsaturated lipids” (Holley et al., (1993)
Br Med Bull 49: 494-505). This degradation typically involves electron gain from the carbon-carbon double bond of the unsaturated lipid, which is a key process in free radical-mediated reactions and subsequent cell death. The first characterization of the 1940s showed that lipid peroxidation was not found only in the symptoms of pathological diseases, but was found in everyday life (eg, "odors" affecting food, polymers and plastics). ) An ubiquitous oxidation process. In biological tissues, lipid peroxidation occurs in cell membranes. The cell membrane structure of the tissue is composed of lipids with a lipid to protein ratio of the order of 1: 1.
Different for each organ. On the other hand, mitochondrial membranes have a somewhat higher protein concentration, about 80%. Most lipids are phospholipids and contain a glycerol major component and a polar terminal region. The non-polar head is a fatty acid, usually 14-20 carbons long, consisting of long chain carbon groups linked by esters. The cis duplex produces a long, straight chain. The more unsaturated the fatty acid, the more suspicious the oxidative attack will be.
Arachidonic acid is a common 20-carbon fatty acid with a double bond at C5, C8, C11, C14 and is a common inflammatory mediator released by cytokines such as prostaglandins. The four double bonds make it a primary target for oxidative attack.

The first step in lipid peroxidation, assuming a normal peroxide release medium, is known as the first chain initial step, which involves the addition of a methylene group (-CH _This is the case where hydrogen ions are extracted from _2- ). As a result, free electrons of the carbon atom (-CH-) remain and similarly become free radicals.
From this, especially in polyunsaturated lipids such as arachidonic acid, a binding density occurs, which remains at a stable end point and free electron species spread through the fatty acid chain until it typically reaches near the end of the chain. As used herein, lipid radical interaction with O ₂ results in a peroxo group (CHO ₂ ), which is then abstracted by other hydrogen ions, resulting in a self-perpetuating autocatalytic reaction. Lipids having a hydroxylated peroxo (peroxyl) group are lipid hydroperoxides, which can be further broken down, react with themselves, yielding cyclic peroxides, which are then broken down into cyclic endoperoxides. The final (stable) end product after the reaction of oxygen with endoperoxide and subsequent hydrolysis yields malondialdehyde (MDA), a three carbon molecule with double-bonded oxygen atoms at both ends. These end products are not uniformly decomposed. For example, arachidonic acid degradation due to oxygen attack produces at least six lipid hydroperoxides and cyclic peroxides and other products.

Although the need for lipid peroxidation is not completely known, metal catalyzed (Fenton) reactions are believed to play a major role. The hydroxyl group readily initiates site-specific hydrogen ion abstraction and promotes continuous peroxide autoxidation. Hydroxyl group formation does not appear to be necessary for the initiation of peroxidation. OH-impurity remover does not hinder this process. Iron is also important in the latter embodiment of lipid oxidation. Lipid peroxide (R-OOH) readily reacts with iron (II) -bound complexes, forming iron (III) -oxide-bound complexes, extra OH ^- and alkoxy (alkoxy) groups (RO-). Occurs. Further, an oxidized ferric iron-complex
) Can also react with lipid peroxides, forming peroxy groups and iron oxide complexes, for example, even at relatively slow rates, thereby essentially reusing the iron. Alkoxy and peroxy radicals abstract hydrogen ions and stimulate lipid peroxidation. These reactions of iron with lipid peroxidation favorably compete with the Haber-Weiss reaction, with K ₂ 76m ⁻¹ s ⁻¹ of hydrogen peroxide being the K ₂ (secondary) of lipid hydroperoxides with iron complexes. (Order rate constant) 1.5 × 10 ³ m ⁻¹ s ⁻¹ .

[0211] The number of iron, including proteins that promote lipid peroxidation, is higher than that used for Fenton hydroxyl formation. The group of iron-binding molecules that stimulate lipid peroxidation includes ATP, carbohydrates, DNA and membrane lipids. This intracellular iron is also used for the Haber-Weiss reaction. In contrast, tightly bound iron-containing molecules that preserve the overwhelming majority or cellular and extracellular iron are not available for the Fenton reaction (if iron is not released) but contribute to the lipid peroxidation reaction. Become. These proteins are ferritin, hemosiderin, lactoferrin, transferrin and heme proteins. The utility of these proteins, especially heme proteins, is directed at the erythrocyte membrane as the primary target of lipid peroxidation. Hemoglobin in red blood cells
It is sequestered by high concentrations of catalase and glutathione reductase and is clearly limited to this type of process.

3.1. d Methods for Measuring Lipid Peroxidation Due to the difficulties of directly measuring lipid peroxidation in vivo, several methods for measuring relative lipid peroxidation have been developed. The most common of these are TBA
RS, or thiobarbituric acid reactive substance assay (Darley-Usmar et al., (199
4) The Biochemist 18: 15-18; Portoles et al., (1993) Biochim Biophys Act
a 1158: 287-92; McKenna et al., (1991) Anal Biochem 196: 443-450; Kosugi
et al., (1994) Biol Pharm Bull 17: 1645-1650; Wallin et al., (1993) Anal
Biochem 208; 10-15; Severn et al., (1993) Eur J immunol 23: 1711-1714;
Augustin et al., (1991) Life Sci 49: 961-968; Vasankari et al., (1995) C
lin Chim Acta 234: 63-69; Sandhu et al., (1992) Free Radic Rec Commun 16
: 111-122; Yagi et al., (1984) Methods Enzymol 105: 328-331). TB
The ARS assay uses thiobarbituric acid, which, when heated under acidic conditions, forms a chromogen whose color intensity at 532 nm is directly proportional to the amount of reactants formed. As measurement of the amount of malondialdehyde (MDA), the end product of lipid peroxidation, has developed from the outset, the TBARS assay also reacts with other substances to form chromogens.
These required adducts include deoxyribose, protein linkages and amino acid compounds. Precipitating proteins and lipids using phosphotungstic acid-sulfuric acid and eliminating or minimizing unwanted reactions by using acetic acid instead of trichloroacetic acid (TCA) to avoid reaction with sialic acid. The extraction condition is TB
Important for performing ARS assays. The TBARS test is susceptible to oxidation and depends on the antioxidant status and serum iron and lipid levels,
Store sample at 0 ° C with important factors. Despite these difficulties, TB
The ARS assay is a direct method of measuring relative lipid peroxidation,
(High performance liquid chromatography) correlates with method (slightly overestimated). TB
Since the ARS test is calibrated with NMA (MDA itself is unstable and therefore hydrolyzes 1,1,3,3-tetramethoxypropane for the actual measurement), the result from the assay is the MDA production It is typically expressed in quantity or simply in absorption units.

3.2 Method 3.2. a Experimental protocol-hemorrhagic shock Male Wistar rats (250-350 gm, Charles River Laboratories,
Inc., Wilmington, Mass.) Were housed in a controlled environment and fed normal small grain feed for at least 3 days before the experiment was started. Animals were pentobarbital (50 mg /
(kg intramuscular injection, Abbott Laboratories, North Chicago, IL), cannulated femoral artery and vein (PE-50 polyurethane tubing, Clay Adams, Parsippany, NJ) and custom made Lucite Put on the table. Heparin was not injected unless it was necessary to ensure an open catheter line (10 U / ml Plasma-Lyte, Upjohn Comp., Kalamazoo, MI). One of the femoral arteries is monitored for mean arterial pressure (MAP) monitoring and pulse pressure (Beckman Instrum).
ents). For rat mesenteric experiments, a central incision is made in the abdomen and the mesentery is carefully placed on an intravital microscope table and handled minimally.

Hemorrhagic shock experiments showed that thin mesenteric preparation did not show reperfusion wounds exposed to an open atmosphere, probably due to ambient oxygen diffusion.
To this end, a specific suitable plastic sheet (3 mm thick) is fitted to the animal tissue specimen and attached to the table using Velcro straps, with small openings and catheter lines on the head and tail. Two other openings serve for displacement and gas injection of the microscope target. Nitrogen gas was injected, and the mesenteric tissue specimen was placed in a hypoxic environment. Mesenteric microcirculation was established using Krebs-Henseleit bicarbonate buffer (118 mM sodium chloride, 4.7 mM potassium chloride, 2.5 mM calcium chloride, 1.2 mM) saturated with 95% N ₂ -5% CO ₂ gas mixture. Magnesium sulfate, 1
. 2 mM potassium, 25 mM sodium bicarbonate, chemicals from Fisher Scientific,
(Obtained from Fair Lawn, NJ) was observed via in vivo fluorescence microscopy.

Fifteen minutes after MAP and plus pressure stabilization, propidium iodide
(PI) (1 μM) (Sigma Chemical Co., St. Louis, MO) was added to the perfusate and background autofluorescence was recorded in selected tissue areas. Next, the first reading was performed in the light area, and the venules and arteries (20 μm-100 μm) were selected and used as fluorescent images. 4-5 observation area is selected at random, and reading is performed every 20 minutes.
CD) Record using digital color of camera (Optronics Engineering; Goleta, CA)
A 40 × water immersion objective (Zeiss; Thornwood, NY) was transferred to a color video monitor (Panasonic CT 1383 VY, Japan) and a cassette recorder (Pa
nasonic, AG-1270, Japan). Images were recorded for later analysis. Fluorescence excitation exposure time was minimized to avoid photobleaching.

After 20 minutes of stabilization time (5 minutes after the addition of PI to the perfusate), hypotension showed a MAP of 4
Induced by a gradual decrease in the volume of blood collected from the femoral artery catheter for 20 minutes to reach 0 mmHg. Thereafter, a small aliquot of blood was removed or heparinized, plasma-lite injected, and MAP maintained at a specified level of hypotension for 100 minutes. The blood volume collected at the time of bleeding and hypotension was at least 3% of body weight. After 100 minutes of hypotension, 37
Warmed again in water bath and slowly returned by intravenous injection over 20 minutes. Blood is collected for serum analysis and an equal or slightly bulky plasma-light (about 2 m
l).

3.2. b Measurement MAP and heart rate were recorded throughout the shock protocol. Arterial samples are collected in heparinized vials at intervals before and after hypotension and reperfusion. A 0.5 ml sample was collected at t = 0 and at 230 minutes (2 hours after reperfusion) for NBT testing. In addition, for selected animals, a 1.75 ml sample was collected at t = 0, 60, 100, 115 minutes (15 minutes after reperfusion) and lipid peroxidation and plasma peroxide were measured at 230 minutes. Samples were centrifuged directly at 1000 G for 30 minutes to separate plasma from cells. The plasma was then stored at -70C. Rat plasma before and after hemorrhagic shock was tested on natural donor leukocytes in whole blood obtained from rats not exposed to hypotension. The reduction of nitroblue tetrazolium by leukocytes resulting from superoxide production was then tested. For the test, 0.1 ml of the donor's whole blood was replaced with 0.4 ml of hemorrhagic shock rat plasma.
And mixed. Anesthesia was induced with pentobarbital (50 mg / kg intramuscular injection) and the femoral artery of the donor animal was cannulated (PE-polyurethane tube). The reconstituted blood mixture was incubated for 10 minutes at 37 ° C. and then subjected to NBT testing. Neutrophil actin polymerization was also measured using the pseudopodia formation test. The test was described in Example 2.

3.2. c Measurement of cell death in mesentery Video tape was replaced for cell death analysis to measure PI fluorescence. Veins were restricted to 20-80 μm in diameter for analysis. The number of PI-positive cells was calculated as the first time point in 4-5 arbitrarily determined mesenteric regions and collected every 20 minutes. The state of the entire area, about 300 μm × 300 μm, was used for this purpose. Many dead (PI positive) endothelial cells were also found in typical vessels. The number of dead cells was compared at different times throughout the experiment.

3.2. d Plasma assay for lipid peroxidation-TBARS assay Plasma lipid peroxidation was collected at regular intervals during hypotension and reperfusion. As described above, a 0.25 ml blood aliquot was collected and centrifuged directly at 1000 G for 30 minutes. Plasma and red blood cells were separated and stored directly at -70 ° C until analysis. In the case of the TBARS assay, a modified method based on Yagi ((1984) Method Enzymol 104: 328-331) was used. In this method, 100 μl of plasma was mixed with 2 ml of N / 12 H ₂ SO ₄ and gently stirred. Next, 0.25 ml of 10% aqueous phosphotungstic acid was added and mixed. The mixture is left for 5 minutes, 30 minutes for 10 minutes.
Centrifuge at 00 rpm. The supernatant was discarded and the precipitate was mixed again with N / 12 H ₂ SO ₄ and 0.15 ml of phosphotungstic acid. This was mixed once more and centrifuged at 3000 rpm for 10 minutes. Next, the supernatant was discarded, and the precipitate was mixed with 1 ml of purified H ₂ O and 1 ml of TBA reagent to prepare an equal volume of an aqueous solution of glacial acetic acid and 0.67% thiobarbituric acid. The resulting mixture was heated in a water bath at 95 ° C. for 60 minutes. After cooling, 2 ml of n-butanol was added to the mixture and stirred vigorously. After centrifugation at 3000 rpm for 10 minutes, the supernatant was analyzed spectrophotometrically (Perkin-
Transferred to a clean plastic vial for Elmer Lambda 3B spectrophotometer). The measurements were performed on distilled water at 532 nm and 600 nm, and the resulting absorbances were subtracted from each other to obtain relative absorbance values. Then, when this absorbance value was inserted into a least square fit calibration curve using a known MDA value, MA
The absorbance for D concentration was obtained (the curve was calibrated with malonaldehyde (MDA), the final product of lipid peroxidation and the absorbance was measured at 532 nm).

3.2.e Plasma Peroxide Assay Plasma peroxide concentrations were determined through the shock protocol at t = 0, 60, 90,
Measured at times of 120 and 230 minutes. Collecting the plasma sample as described above,
The peroxide concentration was measured as described in Example 2, section 2.3.c, but the peroxide concentration measured in the shock protocol was only from plasma and remained in contact with the sedimented cells It has the essential difference that it is not derived from the plasma layer of centrifuged blood. Thus, the measured plasma peroxide in a sample centrifuged at 1000 G for 30 min (500 G for 10 min for normal centrifugation, 10 min) and then frozen at −70 ° C. is not cell-derived (No cells are present) nor is production interrupted by freezing of plasma.

All chemicals were purchased from Sigma Chemicals, St. Louis except where indicated. Results are shown as mean ± SD for all samples. Student's t test
(two-tailed unpaired) was used for all comparisons. Differences with p <0.05 were considered significant.

3.3 Results A representative time course of mean arterial pressure (MAP) of survivors (n = 3) and non-survivors (n = 5) subjected to hemorrhagic shock was performed. 10 minutes later, equilibrated blood was added to 40 mmHg MAP.
Was slowly taken in a step-wise manner until was achieved. 4 continuous during low blood pressure time
Blood was returned or taken as needed to obtain 0 mmHg ± 5 mmHg. During reperfusion, the survivor's blood pressure returned to sustainable pressure, while the non-survivor's blood pressure rose transiently and, finally, despite repeated efforts to restore blood volume and pressure. But fell irreversibly. The results indicate that the mean arterial blood pressure of non-survivors tends not to recover after re-infusion of separated blood, even when adequate volume replacement is provided. This group also appears to require a lower blood pressure injection, a preliminary infusion of blood or Plasma-Lyte. In comparison, MAP in survivors tends to recover to near pre-shock levels after reperfusion.

The application of rat shock plasma (n = 3) to donor rat blood showed a significant increase in NBT (+) neutrophil counts compared to plasma (n = 8) taken from the same rat before the shock protocol. Large rise (P <0.001). Before hemorrhagic shock (t = 0) (n = 8
) And after 100 and 120 minutes of reperfusion (t = 230 minutes) (n = 3), neutrophil activation assessed by the nitroblue tetrazolium test with peroxide production was compared. There was a significant increase in neutrophils as measured by NBT in plasma of shocked rats.

A set of representative images taken using in-vivo microscopy in rat mesenteric preparations was prepared, showing a large increase in PI-positive cells and venule vasoconstriction 20 minutes after reperfusion. Was. DCFH fluorescence (n = 3), however, was not significantly different from control preparations (n = 3) that had been subjected to sham bleeding. Reperfusion after hemorrhagic shock was measured using propidium iodide, with all cells found in the preparation,
The initial values after reperfusion to the end of the experiment resulted in a significant increase in mesenteric cell death in n = 5 visual fields, n = 2 animals, and when compared to endothelial cells alone (p <0. 0
5). The results, measured with propidium iodide until the beginning of the reperfusion time, confirm that there is a slight increase in cell number if there is a sharp increase in the amount of cell death. This increase persists until the end of the experiment.

Endothelial cell death lags generalized (stromal) cell death. Endothelial cell death is generally inconsistent with its preparation, and there is a delay of about one hour after parenchymal cell death before significant endothelial cell death. This finding was surprising in light of the fact that endothelial cells were not only exposed early to circulating toxins, but were also a major producer of free radicals in shock. Comparison between the two plots of each graph shows evidence for a contribution of increased neutrophil "preactivation" levels to cell damage.

Another experiment uses the peroxide electrode method to measure the time course of peroxide formation in plasma peroxide concentrations in rats subjected to hemorrhagic shock, as measured ex vivo. A small rise in plasma peroxide was recorded during the hypotension period, followed by a major jump after reperfusion of flowing blood. Levels are low until late in the hypotensive period if there is a rise in peroxide concentration immediately after reperfusion, followed by a sharp rise in plasma. The overall levels recorded are below normal levels due to a variation of the test using frozen plasma in the absence of cells rather than the plasma layer of centrifuged blood. The time course of lipid peroxidation during global ischemia and subsequent reperfusion time is determined by measuring the lipid peroxidation TB
The time course of ARS production was measured by measuring absorbance at 532 nm during hemorrhagic shock. The level increased before reperfusion of flowing blood, but suddenly increased after reperfusion,
Subsequently decreased. As in cell death measurements and plasma peroxide measurements, there is a slow increase in plasma TBARS concentration throughout the hypotensive period. The concentration increases substantially in the reperfusion layer.

3.4 Discussion Increased levels of neutrophil "pre-activation" correlate with increased lethality in hemorrhagic shock (Barroso-Aranda et al. (1992) Circ Shock 36: 185-190; Barroso -Arand
a et al. (1989) Am J Physiol: H846-852; and Shen et al. (1990) Circulator
y Shock 31: 343-344). Although "pre-activation" is observed in these cases, there is currently little understanding of the mechanisms underlying the increased lethality observed in animals with increased levels of "pre-activator". The observation that activators occur in circulating plasma indicates that "pre-activation" is a systemic phenomenon. Thus, "pre-activation" can lead to some forms of organ failure and may be responsible for up-regulating certain autoimmune and host defense responses over time.

“Free radical theory of toxicity” is supported by compelled evidence implicated as a lethal mechanism in systemic ischemia / reperfusion. If elevated levels of "preactivators" result in increased lethality in global ischemia / reperfusion, one mechanism for this increase in lethality is upregulation of the oxygen free radical production system, followed by excess toxic free radical species. It seems to be due to production. It is known that membrane degradation by either free radical oxidation or other factors can form biologically active mediators that activate neutrophils, among other functions. The dominant among these neutrophil activating lipids is platelet activating factor (PAF) or a PAF-like substance, which is formed via oxidative damage of the cell membrane (see also Example 9).

Hemorrhagic shock (Wiggers' model) appears to involve up-regulation of neutrophils and other cells, free radical interactions, lipid peroxidation, cell dysfunction and eventual organ death A well-studied but incompletely understood model of acute trauma. While there is undoubted synergy between these events, the relative importance of these various entities and the course of activation over time is unclear. Cell death in skeletal muscle due to hemorrhagic shock, visualized by propidium iodide, increases before overt leukocyte accumulation due to endothelium-derived free radical production. This, however, does not rule out the possible major role of neutrophils in other organs (notably in the visceral area and lungs), which are subsequently circulating mediators that ultimately affect more robust skeletal muscle Can be produced.

The results presented here support the conclusion that neutrophil activation is increased after hemorrhagic shock, resulting in increased plasma peroxide production, which can subsequently affect lipid peroxidation and cell viability. I do. A linear interrelationship of the degree of neutrophil activation present before the shock step and after reperfusion and the relative amount of plasma lipid peroxidation detected was observed.
This reciprocal system indicates that the lipid peroxidation detected here is not just a passive by-product of cellular free radical attack and subsequent cell death, but is a major component of neutrophil activation and possible "pre-activation". This is one of the reasons. A correlation was observed between the degree of "pre-activation" as assessed by NBT (+) superoxide production and lipid peroxidation as measured by TBARS. The results show a significant degree of interactivity between NBT (+) activation before (r2 = 0.992) and after (r2 = 0.838) shock, and the relationship between the two events Show the possibility.

The discovery of increased levels of neutrophil activators in unprotected tissues such as the mesentery and in the form of “pre-activation” prior to cell death (see above) is due to circulating neutrophils. Turn activators into significant components of the shock process.

Example 4 Visceral Arterial Occlusion Shock and Plasma Activation in Rats Summary The link between visceral artery occlusive shock (SAO) shock and the presence of neutrophil activator in plasma from SAO shocked animals is described in this example. Describe. Rats were randomly divided into shock and sham shock groups. Abdominal wall incisions were made in the shocked animals to occlude the upper mesentery and celiac arteries. After 90 minutes, the ligation was removed and the visceral area was reperfused. SAO shock was evidenced by a sharp decrease in systemic blood pressure during reperfusion. Aliquots of blood were taken before occlusion and after reperfusion, and neutrophil pseudopodia formation, an indicator of NBT activity and leukocyte activation, was measured.

The results show a significant increase (p <0.001) in the activation of leukocytes by plasma from SAO shocked animals in both sets of assays. Plasma from sham-shock rats did not show increased activation. These results indicate that plasma activation is
It demonstrates that it occurs with AO shock and that fluid activating factors that can be co-located with myocardial depressors found in the pancreas are derived from visceral areas, particularly the gastrointestinal tract and pancreas.

4.1 Introduction As shown in Example 3, there is a strong cardiovascular "activator" in the plasma of animals subjected to hemorrhagic and endotoxin shock, the presence of which is Not only does shock status increase significantly, but also correlates with decreased survival in models of hemorrhagic shock. However, it was unknown whether visceral artery occlusion (SAO) shock induced up-regulation of leukocyte activation. This form of ischemia / reperfusion injury is important because it isolates the visceral region as a possible precursor moiety for activator formation. The discovery of neutrophil activation in such shock models may lead to insights into the origin of neutrophil activators. This study was designed to determine whether visceral arterial occlusive shock induces cardiovascular cell activation.

[0235] SAO shock is a form of shock that involves a visceral region by ligating one or more major supply arteries in this region. The main artery supplied to the visceral area
The superior mesenteric artery, which comes directly from the aorta and is supplied to the mesentery of the pancreas, duodenum and small intestine. This vascular occlusion results in consistent lethality in dogs within 12-48 hours. One of the salient features of this model is that occlusion of the superior mesenteric artery is often fatal, even before the bowel loses its viability. In addition, release of the obstruction leads to death more reliably and faster than if the tissue is maintaining ischemia. The latter observation indicates the sensitivity of the visceral region to "reperfusion injury" from either free radical interactions or other circulating toxic metabolites. If only the upper mesenteric artery is occluded due to the side-by-side flow from other blood vessels, especially the celiac and lower mesenteric arteries, most of the intestine will remain alive and for several hours, Most leave viability.

The model of visceral artery occlusion shock used in this experiment involves ligation of the superior mesenteric and celiac arteries. The celiac artery provides side-by-side flow to the superior visceral area (such as the pancreas), and ischemia in both arteries leads to a faster and more consistent appearance of lethality than occlusion of the superior mesenteric artery alone. Bring. The third major supply vessel to the visceral area, the inferior mesenteric artery, can also be ligated, which leads to necrosis of the large intestine and intestine which is undesirable in this experiment due to the potential for bacterial migration. . Ligation of the superior mesentery and celiac arteries guarantees almost complete ischemia of the pancreas, but the large intestine remains relatively well perfused. This SAO shock model is well studied and relatively reproducible.

SAO shock, an established model with more localized areas of tissue exposed to ischemia / reperfusion, measures whether circulating neutrophil activators are regenerated in visceral areas Selected for. Ischemia / reperfusion of the visceral area results in release of myocardial depressant factor (MDF). Upon reperfusion, MDF circulates and suppresses cardiac contraction, resulting in impaired cardiac function, decreased blood pressure and exacerbated shock. It has been recognized that this factor is partially released from the pancreas, is sensitively sensitive to low flow conditions, and is partially sensitive to diversion of blood flow to more “critical” organs (heart, brain). It easily becomes ischemic in a flowing state. Thus, the development of global hypotension or direct ischemia of this organ results in the release of MDF. The circulating neutrophil activator found during hemorrhagic shock also increases
It is also made with AO shock, and may be co-located with MDF, or perhaps even identical.

4.2 Methods Male Wistar rats (250-350 gm, Charles, River Laboratories, Inc.
Wilmington, MA) was maintained in a controlled environment and maintained on a standard pellet diet for at least 3 days before the start of the experiment. Rats were randomly divided into SAO shock (n = 10) and sham SAO shock groups (n = 11). The femoral artery and vein of the animal were cannulated under general anesthesia using pentobarbital (50 mh / kg im, Abbot Laboratories, North Chicago, II) (PE-50 polyurethane tubing, Clay Ada
ms, Parcippany, NJ). Heparin was not infused except to confirm that the catheter line was open (10 U / ml Plasma-Lyte, Upjohn Comp.,
Kalamazoo, MI). The femoral artery was cannulated and mean arterial pressure (MAP) and pulse pressure were monitored (Beckman Instruments, IL). A central incision was then made in the abdomen. The superior mesentery and celiac artery were isolated. SAO shock was induced by total ligation of these arteries. In sham (control) animals, the artery was isolated but not ligated. The abdominal cavity was closed and covered with a layer of gauze soaked in warm saline. At the end of the immersion time (90 minutes), the central incision was opened again and the ligation was removed in the group of shocked animals. Arterial blood samples before and after the SAO shock, during the SAO shock and in the sham group were collected in heparinized vials. Immediately sample for 30 minutes, 10
The plasma was separated from blood cells by centrifugation at 00G. Plasma for further analysis -7
Stored at 0 ° C.

The plasma of rats before and after SAO shock and in sham control rats was tested against whole blood, untreated donor leukocytes from rats not exposed to shock. Nitroblue tetrazolium reduction by leukocytes due to superoxide production was measured. For such tests, 0.1 ml of donor whole blood was mixed with 0.4 ml plasma from SAO shock and sham SAO shock rats. Donor animals were anesthetized with pentobarbital (50 mg / kg im) and cannulated into the femoral artery. Incubating untreated control blood with experimental plasma for 10 minutes at 37 ° C.
Then, it was subjected to the NBT test described in Example 2. NBT reduction is due to superoxide formation and can be blocked with superoxide dismutase (SOD).

Measurement of human neutrophil pseudopodia formation by application of shock plasma was also performed as described in Example 2. Pseudopodia formation is due to actin polymerization, another indicator of neutrophil activation.

For the measurement of myocardial depressants in shock sera, stock samples of SAO shock and sham SAO shock sera were filtered at 1300 G and 3000 kD filter.
(Centricon, Amicon Corp., Becerly, MA)
The filtrate from 0 (mean number of animals per pool = 6) was placed in random numbered vials on dry ice for analysis of myocardial depressant factor (MDF) and the Krebs-Henseleit solution I sent it to various places along with the control. Samples were assayed for suppressor activity in electrically driven isolated papillary muscle from the right ventricle of the cat heart. The developed tone of the isolated papillary muscles was recorded on an oscillograph recorder. MDF formation was recorded as the absolute value of the rate of change in cat papillary muscle contraction under standard conditions of 37 ° C. and 1 Hz frequency as reported in (Lefler (1970) Clrc Res 26: 59-69).

4.3 Results A 90-minute occlusion of the superior mesenteric and celiac arteries is a reproducible model of intestinal shock, with consistent lethality of rats (n = 16) within 2 hours of ligation removal. Bring.
Shock was characterized by an immediate rise in blood pressure of about 6-12 mm Hg when the artery was ligated. Mean arterial blood pressure (MAP) remained elevated throughout the experiment, and the animal appeared to maintain normal heart and lung activity until the ligation was removed, at which time the blood pressure dropped sharply to about 40 mm Hg ( P <0.05 compared to the sham shock group (n = 4). This pressure is then typically reduced to about 10 until the animal gradually dies.
Decreased from minutes to about 2 hours. The mean arterial pressure of the SAO shock group and sham shock of rats subjected to the method was measured. An increase in blood pressure when the artery of the shocked animal was occluded and a sharp decrease when the ligation was removed were observed. Sham shock animals did not show significant changes in blood pressure. The mortality was 100% in 2 hours.

SAO shock was measured by the formation of neutrophil activator in plasma (P <0.001 compared to sham shock group and shocked animals before shock protocol) and pseudopodia formation as measured by NBT test. (P <0.001 compared to both the sham shock group and the shocked animals before the shock protocol). S
The percentage of neutrophils showing pseudopodia from donor blood induced by plasma before AO shock and sham (first) shock and after (final) shock was measured.

There was no significant enhancement of neutrophil activation measured in control animals by either assay during the course of ischemia and reperfusion. Myocardial inhibitory activity was determined by comparing SAO shock animals to sham SAO shock animals compared to control plasma samples.
Comparison of (compared to pseudo shock animals ^* P = 0.065) myocardial depressant factor in (MDF) activity was increased in all SAO samples except one (P = 0.065).

4.4 Discussion This experiment shows that SAO shock activates neutrophils as measured by NBT test and pseudopodia activation. Leukocyte activation has been previously observed in other shock models, but its presence is not equivalent to SAO shock. The finding that the activating factor is found in plasma indicates that this neutrophil activation is a systemic phenomenon, and the finding of the factor in SAO shock indicates visceral as a potential organ.

The identity and mechanism of neutrophil activation released by SAO shock is unknown. Interleukin-2 (Il-2) and tumor necrosis factor-α (TNF-α)
And different mediators such as monocytes and lymphocytes have been reported to be activated during SAO shock. Platelet activating factor (PAF), phospholipids released from damaged cell membranes or reported to be involved in this shock model,
Inhibition of AF has been shown to be beneficial in SAO shock. (The role of PAF as a possible neutrophil activator is detailed in Example 9.)

Although the factors that activate neutrophils in SAO shock have not been finalized, similar to hemorrhagic shock, the etiology seen in this shock model is due to leukocyte upregulation and inhibition of this activation. Involvement and showing that it may have protective effects systemically and systemically. SAO shock is an ischemia / reperfusion injury model that primarily targets the pancreas by ligating its two major supply arteries. Ischemia / reperfusion experiments are also performed on the pancreas alone, where the arteries supplying the pancreas (gastroduodenum, spleen, gastrica sinstra and gastricae breves (gas
tricae breves)). Ligation of these arteries for 60 minutes induces acute pancreatitis with significant neutrophil accumulation, but no fatality has been reported after 120 minutes. This is consistent with the results of the experiments presented herein that a 60 minute SAO shock was not necessarily lethal to rats within a 2 hour reperfusion time (data not shown).
In pancreatic ischemia / reperfusion injury, like SAO shock, the "trigger event" of organ injury and subsequent death appears to be a free radical burst initiated by reperfusion of the pancreatic and intestinal regions, which then It leads to the activation of leukocytes, endothelial cells and the subsequent release of lipids such as Dietia and pancreatic proteases. Inhibition of free radicals by superoxide dismutase (SOD) and catalase attenuates this damage, in part, by reducing PAF production by oxygen free radicals.

Alternatively, the acute sequelae that occur after loosening the ligation of arteries is not a free radical mediated event, primarily due to sudden reoxidation of tissue, but rather a sudden release of proteases and other toxic components from the pancreas into the general circulation. Is caused by the release of For example, in phenomena such as the influx of leukocytes into the pancreas, pancreatic edema, and inflammation, indicate that flow does not occur until the flow is reestablished outside the circulation. Apart from the trigger mechanism, hypoxia and the release of toxic mediators of the pancreas due to complete ischemia can be lethal to the organism.

[0249] SAO shock generally involves selective ischemia / targeting of the visceral and specific pancreas.
Reperfusion injury. This form of injury results in up-regulation of systemic cardiovascular cell activators and other harmful mediators such as myocardial depressants. These mediators originate in the pancreas and may be more inhibitors of pancreatic damage than ischemia / reperfusion itself.

Example 5 Localization of Neutrophil Activator in Tissue Summary Hemorrhagic and endotoxin shock and shock following visceral artery occlusion (SAO) result in up-regulated levels of leukocyte activation. Activation results in pseudopodia formation and nitroblue tetrazolium (NBT) testing in donor neutrophils exposed to shock plasma. To determine the origin of the responsible factor, homogenates were made in rat organs, which were then tested for activation of untreated neutrophils. Randomly selected rats were weighed, anesthetized, and arterial and venous catheters were inserted. An abdominal wall incision was made and the animals were bled whole. Organs including spleen, small intestine, pancreas, heart and liver were immediately removed and placed in 0.25M sucrose solution for homogenate. In two animals, no kidney and adrenal glands were collected. Then the organs were 1: 3
(w / v) Homogenized in Krebs-Henseleit solution. After homogenization, the suspension was then further diluted with 1: 2 (v / v) Krebs-Henseleit.
Each homogenate was divided into two equal parts: one stored at 4 ° C and the other incubated for 2.5 hours at 38 ° C to determine if endogenous tissue enzyme activity promotes activator release. did. Both sets of samples were tested for neutrophil pseudopodia formation and NBT activity. The results showed a significant increase (P <0.001) in leukocyte activation by the incubated pancreatic homogenate and a small but significant (P <0.005) increase in the unincubated homogenate. Activation from all other organs was a nonsignificant increase compared to control samples.

The activation occurring in pancreatic homogenates of other species was then measured. Five boars were randomly selected, the pancreas was removed and placed in a 0.25M sucrose-saline solution for homogenization. Organs were homogenized in 1: 4 (w / v) saline solution.
Samples were incubated for 2.5 hours at 38 ° C. and tested for neutrophil pseudopodia formation and NBT activity. The results of both studies show a significant (P <0.001) increase in leukocyte activation by the incubated porcine pancreatic homogenate as compared to the control.

Demonstration of rat and porcine pancreas homogenate activator size. Aliquots of rat and pig pancreatic homogenates were ultrafiltered using a 3 kD cutoff. Unfiltered and low molecular weight fractions activate neutrophils (P <0.001)
), Indicating that at least one of the in vitro neutrophil activators of rat and pig pancreatic homogenates is a low molecular weight species. The results indicate that the pancreas acts as an endogenous source of neutrophil activators in shock and inflammatory conditions.

5.1 Introduction The visceral region is implicated as a potential precursor for neutrophil activator thermogenesis (PMS) (Example 4). Because systemic hypotension, endotoxin shock and SAO shock are involved in ischemia in the visceral area, low flow in this area may be a common mechanism leading to the formation of circulating neutrophil activators in shock.

In addition, other investigators have identified, in addition to other models, myocardial inhibitory factors (MDFs) that suppress cardiac contractility and are released by hemorrhagic, endotoxin, and visceral arterial occlusive shocks. (Glenn (1971) Circ Res 29: 238-249). The major difficulty in isolating and identifying this compound is obtaining a sufficient amount of material for the assay. Lefer et al. (
For example, Lefer (1970) Am J Physiol 218: 1423-1427; Lefer (1972) Am J Physiol
ol 223: 1103-1109) tested homogenates from different organs and found that after mild incubation, it was possible to produce MDF ex vivo.

Because of the similar potential between the production of MDF and the neutrophil activator isolated herein, for the spatial identification of the source of the neutrophil activator and this Due to the determination of whether such factors are endogenous (performed in tissues as opposed to being synthesized during the state of shock), neutrophil activation seen in shocked plasma is not The question was whether the homogenate would regenerate neutrophils to activate them ex vivo.

Anesthetize tissue samples of small intestine, spleen, pancreas and liver, heart, kidney and adrenal gland,
All blood samples were taken, homogenized and incubated. They were then tested for their ability to activate donor neutrophils using the NBT and pseudopodia formation tests. The porcine pancreas was tested to determine if organ-induced neutrophil activation was a species-dependent phenomenon. To identify the molecular size of these neutrophil activators, pancreatic homogenates were ultrafiltered through a 3 kD filter and assayed for activation.

5.2 Methods The organ homogenization method is described in Lefer (Glenn et al. (1971) Circ Res 29: 338-349.
, Lefer (1973) Am J. Physiol 224: 824-831). Male Wistar rats (250-350 gm) were housed in a controlled environment and maintained on a standard pellet diet for at least 3 days before the start of the experiment. The femoral artery and vein of the animal were cannulated under general anesthesia using pentobarbital (50 mg / kg im). No heparin was injected (10 U / ml Plasma-Lyte) except that it was necessary to check that the catheter line was open. A central incision was made in the abdomen. Rats were bled and the heart, liver, spleen, small intestine and adrenal gland were removed. In two animals, kidneys and adrenal glands were similarly removed. They were washed immediately and cleaned with cold 0.25M sucrose solution. The cleaned organs were vigorously homogenized (1: 3 w / v) in Krebs-Henseleit solution. The homogenate was then further diluted with Krebs-Henseleit solution (1: 2 v / v). Aliquots were filtered by centrifugation at 500 G for 10 minutes and samples were stored at 4 ° C. until a constant amount was assayed. The other fraction was incubated for 2.5 hours at 38 ° C. with gentle agitation and assayed. Stored at 4 ° C. Incubation of the tissue homogenate at moderate temperatures for this time promotes enzyme activation, which may be necessary to produce a neutrophil activated product. Incubated and unincubated organ homogenates and controls were tested for pseudopodia formation on untreated human donor leukocytes and tested in the NBT assay in whole rat donor blood. Pseudopodia measurements were performed as described in Example 2 using isolated neutrophils in D-PAS combined with the homogenate at a ratio of 4: 1. Reduction of nitroblue tetrazolium by superoxide in the rat homogenate assay was also tested using the NBT method described in Example 2 by applying 25 μl of homogenate to donor blood. The NBT assay for porcine homogenate activity was also determined by crystal violet staining, using the NBT method described in Example 2 with minor modifications.

To harvest the porcine pancreas, 3-4 months old male Hampshire pigs weighing 18-20 kg were used. Animals were prohibited from feeding 24 hours prior to surgery. Surgical anesthesia was induced with catamine (33 mg / kg / IM), atropine (0.05 mg / kg / IM) and sodium thiopental (10 mg / kg / IV) and with a combination of 1-2% halothane and oxygen. Maintained. Animals were sacrificed with pentobarbital (120 mg / kg / IV) and the pancreas was harvested immediately and rinsed with cold saline.

The pancreas was transferred to a 1: 1 saline: 0.25 M sucrose solution on ice. The pancreatic fat and excess tissue were removed, weighed for 5 minutes and mixed in a commercial blender for 5 minutes using a 1: 4 w / v ratio using saline. The mixed homogenate was shaken vigorously and incubated for 2.5 hours at 38 ° C with shaking every 15 minutes. The incubated homogenate was centrifuged at 800 G for 30 minutes, and the supernatant was passed through a 0.78 μm vacuum filter (Millipore Filter Co., Beberly, Mass.). Pseudopodia formation was measured in human, human donor neutrophils using isolated neutrophils in D-PAS in a 4: 1 ratio with the homogenate using the method described in Example 2. .

An aliquot of the sample was randomly treated with a standard cell culture combination antibiotic-antimycotic agent to break up potential sample contamination and nonspecific activation by potential bacterial products. (Antibiotics-antimycotics (100x) were dissolved in 0.85% saline at 10,000 U / ml penicillin (base), 10,000 μg / ml streptomycin (base), 25 μg amphotericin B (Gibco BRL catalog # 15240- 013, Gibco, Grand Is
land, NY) at a concentration of 0.5% by volume). It has been reported that drug concentrations in this range do not affect neutrophil function.

To test for potential low molecular weight activity in rat pancreas, homogenates were immobilized on a fixed rotor Amicon filter (Model S-100, Centricon, Millipore Filte).
r Co., Beberly, MA) with a 100 kD MW cutoff. Aliquots were saved for pseudopodia formation measurements, and then the filter effluent was also filtered with a fixed rotor Amicon filter (Model S-30, Centricon, Millipore Filter Co., Bebe
(rly, MA) using a 3 kD MV cutoff. The unfiltered precipitate was brought to its original volume with Krebs-Henseleit solution. Keep at 4 ° C. until all ultrafiltration aliquots were used. Measurement of neutrophil activation was performed as described above. In subsequent experiments, the 100 kD filtration step was omitted.

The low molecular weight activity in the porcine homogenate was treated similarly, omitting the 100 kD filtration step. The low molecular weight composition of the homogenate was randomly identified by MALDI mass spectrum
(Mass Spectroscopy Laboratory, The Scripps Institute and Research Founda
tion, La Jolla, CA). Results are shown as mean ± SD for all samples. Student t test (two-tai
led unpaired) was used for all comparisons. Differences with p <0.05 were considered significant.

5.3 Results 5.3.a Rat Homogenate Results Isolated untreated human neutrophils incubated for 10 min with filtered homogenate from different rat organs (n for each organ except n = 2 kidney and adrenal gland) = 6) indicates only a small amount of untreated neutrophils, as measured by pseudopodia activation, except for pancreatic homogenates which significantly (P <0.001 compared to control and all other organs) activate untreated neutrophils. Activation. Intestinal homogenate was not measured in this particular test due to non-specific contamination of the sample.

Incubation of these same tissues (each organ n = 6) for 2.5 hours increases the level of neutrophil activation to 97.2 ± 3.4% in pancreatic homogenates (control and Compared to all other organs, P <0.001), neutrophil activation by other tissue homogenates was not significantly different from control or unincubated homogenate levels.

The NBT assay for superoxide production (n = 5 for each organ) was also significantly (P <0.05 compared to control) neutrophil superoxide production (42.8 ± 1
(5%) activated pancreatic homogenate and also resulted in significant (P <0.05 compared to control) superoxide formation in this assay (20.3 ±
4%), except for the incubated liver homogenate, which resulted in minimal activation of neutrophils in rat whole blood mixed with the incubated organ homogenate. Pancreatic homogenates also induced significantly higher activation when compared to other organ homogenates, and NBT levels in response to incubated liver homogenates were not significantly different from other organ homogenates. Due to the very low levels of neutrophil pseudopodia activating activity (below control values), kidney and adrenal homogenates were not assayed for NBT superoxide production.

Neutrophil pseudopodia formation after incubation in ultrafiltered pancreatic homogenate with a molecular weight of less than 3,000 D (as confirmed by mass spectrum) was also a control value.
(P <0.001) resulted in a significant level of activation (57.6 ± 10.8%) as compared to (6.2 ± 1.5%), but compared to the non-ultrafiltered homogenate. Activity was reduced to some extent. The low molecular weight (<3 kD), medium molecular weight fraction (<100 kD) (60.8 ± 13%) and whole pancreas homogenate fraction (68 ± 8.
9%). The high molecular weight fraction (MW> 100 kD) had significantly lower activation (P <0.05) compared to whole pancreatic homogenate (41.7 ± 8.
8%).

5.3.b Pig Homogenate Results The results of the pseudopodia assay after incubation with porcine pancreatic homogenate (n = 5) show that the low molecular weight fraction (<3 kD) and the unisolated porcine pancreatic homogenate are human isolated Spheres were shown to be significantly activated (P <0.001 in both tests compared to controls). The porcine pancreatic homogenate neutrophil activation rate was 86.2 ± 5.5% and the low molecular weight homogenate was 41.7 ± 19.6%. The increase in neutrophil activation by the low molecular weight fraction is also significantly lower than in all homogenate samples (P <0.05), and that the low molecular weight fraction is probably not a more potent actin polymerization activator than its rat counterpart. Is shown.

Pig homogenate, low molecular weight (<3 kD) and total homogenate also significantly activate neutrophils as assayed in the NBT test using crystal violet staining for cell identification (control in both tests). P <0.001). In this test, there was no significant difference in neutrophil activation by total and low molecular weight samples. As described in Example 2, when comparing the results of an NBT test using this stain with an NBT test using a standard protocol (Wright stain), crystal violet staining provided significantly higher contrast and NBT plasma Care must be taken to facilitate visualization of

Addition of a 0.05-10% vol / vol concentration range of antibiotic-antimycotic agents, as measured by pseudopodia formation, neutrophil activation following normal neutrophil activation or exposure to pancreatic homogenate Does not affect sphere activation. At high doses (100 × of recommended concentration), there is a small dose-dependent activation.

5.4 Discussion This experiment was designed to determine whether neutrophil activators are produced in selected tissues in the body. Such factors are released into the circulation during shock or other traumatic conditions and contribute to leukocyte activation and subsequent systemic damage. Although not tested here, the same activator can also affect other cells of the cardiovascular system (see Example 2 for in vivo observations). Cellular activators are probably
It was hypothesized to be produced by tissue damage in response to oxygen free radicals. Examples of these include platelet activating factor (PAF), tumor necrosis factor-α (TNF-α) and other cytokines. However, it was not finally proven that these known activators were responsible for the first neutrophil activation seen in vivo. It was interesting to determine whether such factors are endogenous to one tissue or formed entirely by ubiquitous cell types such as endothelium and macrophages. Cytokines and bioactive lipids (eg, PAF) can be produced in many organs, but can be specific for certain cell types.

The results provided herein demonstrate a particular role of one organ, the pancreas, in the production of neutrophil activators. In view of the finding that neutrophil activators circulate during shock following visceral artery occlusion (see Example 4) and in response to hemorrhagic and endotoxin shock, a special study of these experiments on organs in the visceral area was made. Attention was paid. These organs are known to be susceptible to manifestations of ischemia and other shocks (eg, acidosis, bacterial migration). The pancreas has been shown to be particularly susceptible to global ischemia, such as hemorrhagic shock, as well as local. Ischemia of this organ results in the production and release of circulatory activators.

As noted above, others (Lefer et al. (1973) Am J. Physiol 224: 824-831, Lefe
r et al. (1970) Circ Res 26: 59-59) have isolated a myocardial inhibitory factor (MDF).
MDF may be a neutrophil activator since the current results of the SAO shock experiment demonstrate the presence of MDF and neutrophil activation (see Example 4). Even though MDF is not a neutrophil activator, endogenous neutrophil activators can be produced or even co-located with MDF. This hypothesis is unlikely.

MDF is postulated to be a peptide bound to long chain fatty acids and has an estimated molecular weight of 800-
It has 1,000 daltons, which is a potential low molecular weight neutrophil activator. Contrary to the results reported herein that even non-incubated pancreatic homogenates strongly activate neutrophils (albeit to a lesser extent than incubated homogenates), little MDF was present without homogenate incubation. Only, indicating that MDF is essentially absent, but is formed by an enzymatic degradation process.

In comparison, the production of pancreatic neutrophil activator provided herein does not appear to be highly dependent on enzyme function. The implication of this finding is that pancreatic neutrophil activator is a preformed molecule released by cell rupture or by ischemia, or formed during shock apart from enzymatic processes. The latter hypothesis is
We exclude small pancreatic peptides from possible neutrophil activators because they tend to be degradation products formed from large (proenzyme) amino acid chains. Alternatively, preformed or small lipids released in response to oxidative stress, cell rupture, or ischemia may function as pancreatic neutrophil activators.

The results of actin polymerization and NBT reduction (see also Example 9 for neutrophil superoxide production results obtained with the chemiluminescence method) are consistent, leading to in vitro neutrophil activation in rats. Identify the pancreas as the only source of homogenate tissue. This finding is surprising because the gut and other gastrointestinal organs involved in neutrophil activation in vivo have little neutrophil activation.

Another interpretation of these results is that while all organs contain factors capable of activating neutrophils, the pancreas is the only neutrophil inhibitory factor among the organs tested herein. That is. The pancreas, the main organ of exocrine and digestive enzymes in the body, is somewhat unique among other organs and may be the site of the enzymatic digestion process. Interestingly, but other studies have shown that homogenates from certain tissues, including the thymus, intestine, spleen and heart,
It was found to be free of neutrophil inhibitors that substantially reduced neutrophil activation compared to controls in a dose dependent manner. No cases where the pancreas was analyzed have been reported. Inhibition has been confirmed in the following experiment (Example 7).

The results of the neutrophil activation test in porcine pancreas are consistent with those obtained in rat pancreatic experiments. Pseudopodia formation and neutrophil testing in neutrophils incubated with porcine pancreas result in significant levels of activation compared to control samples. But,
Inconsistent results with low molecular weight pig homogenate. The low molecular weight pseudopodia formation test shows significantly higher actin polymerization activity than control samples and significantly lower than whole porcine pancreatic homogenate. Rat and pig studies are in agreement.
NBT tests, however, show equivalent activation of neutrophils incubated with either low molecular weight or whole porcine pancreatic homogenates. These findings are consistent with the results of neutrophil lucigenin-promoted superoxide production (see Example 6), which demonstrates equivalent activation of the total and low molecular weight fractions. The reason for this contradiction is unknown.
As described in Example 2, different types of neutrophil activation such as pseudopodia formation, superoxide production and ligand divergence generally tend to segregate. The low molecular weight pancreatic neutrophil activator present in pigs should predominantly activate the NADPH oxidase pathway and, more weakly, actin polymerization.

The results of antibacterial-antimycotic administration show that the presence of these compounds at constant concentrations has little effect on either control or activated samples. Due to the fact that it is almost impossible to make truly sterile homogenates, especially in pigs, administration of antibacterial-antifungal agents as a preventive measure is a prophylactic measure against bacterial contamination and nonspecific neutrophil activation. Can work. Bacteria cannot be filtered through low molecular weight cut-off filters in the tested samples and do not produce a peak in the mass spectrum corresponding to the bacterial chemotactic peptide fMLP.

This experiment serves to discover endogenous in vitro neutrophil activators that develop in pancreatic homogenates and are not in homogenates of other organs. The discovery of porcine pancreatic neutrophil activator analogs indicates that pancreatic neutrophil activator may not be species-specific in rats. Pancreatic neutrophil activators also appear to be formed in various animal species, including humans, because the related low molecular weight substance MDF produced in the pancreas occurs in humans and many laboratory animals.

The results of the experiment indicate that at least one low molecular weight neutrophil activation is emitted from the pancreas, but does not preclude the presence of high molecular weight (20-40 kD) activators like other proteases. Show. The pancreas is a unique organ in that it has a wide range of digestive enzymes and potentially other inflammatory compounds. There may be synergy between whole pancreas homogenate and large proteases and low molecular weight activators.

Example 6 In Vitro Chemiluminescent Measurement of Plasma Superoxide Production by Pancreatic Activator Summary Activators of circulating blood cells are currently poorly defined in shock. As shown herein, pancreatic homogenate, but not the other studied organs (heart, liver, intestine, adrenal gland, kidney), activates untreated donor neutrophils as measured by pseudopodia formation.

Whether neutrophil activation for superoxide production is detected with lucigenin chemiluminescence and whether chemiluminescence is detected from pancreatic homogenates of other animal species, in this case pigs Experimented. The pancreas of six rats was homogenized in 1: 9 (w / v) Krebs-Henseleit buffer, incubated at 38 ° C for 2-5 hours, and aliquots were filtered with a 3 kD cutoff. In a similar experiment, the pancreas was removed from five boars and placed in a 2.5 M sucrose-saline solution for homogenate. Organs were homogenized in a 1: 4 (w / v) saline solution. The samples were incubated for 2.5 hours at 38 ° C., aliquots were filtered with a 300 MW cutoff and tested for chemiluminescence. The samples were measured for superoxide chemiluminescence in human donor plasma. Rat homogenate results show a significant (P <0.001) increase in superoxide-induced chemiluminescence by pancreatic homogenate, unfiltered and low molecular weight fractions. The results of the pig experiment also show a significant (P <0.001) increase in superoxide-induced chemiluminescence by pancreatic homogenate, unfiltered and low molecular weight fractions. These results indicate that porcine pancreas, like rat pancreas, contains factors that activate neutrophils in vitro, including low molecular weight activators.

In other experiments, chemiluminescence of isolated human neutrophils was measured at various concentrations of autogolous plasma, with or without the addition of pancreatic homogenate and known fMLP and PAF stimulants. The results show a transient and spatially distinct superoxide production due to their activation and plasma buffering effects. Pancreas homogenate concentrations used, induced a large superoxide response than fMLP (10 ^-6 M) or PAF (1 0 ^-6 M).

The purified pancreatic homogenate activated other cell types in addition to neutrophils in vitro. For experiments of this possibility, chemiluminescent tests were performed using pancreatic homogenates, low molecular weight pancreatic homogenates or bovine arterial endothelial cells (BAEC) plated on control solution. The results show a significant increase (P <0.001) in chemiluminescence in BAEC cultures incubated with whole pancreatic homogenate. Low molecular weight pancreatic homogenate-induced activation was not significantly greater than control values. These results indicate that the pancreatic homogenate contains factors that activate endothelial cells in vitro. Pancreatic homogenate factors can be potent endogenous activators of neutrophils and endothelium in inflammatory conditions.

6.1 Introduction There are endogenous factors that can upregulate neutrophils in vitro and these factors are present in the pancreas but not in other tissues (see Example 5). Factors can upregulate neutrophils as measured in vitro by pseudopodia formation and the NBT test. Since lucigenin chemiluminescence was used in assays for superoxide formation in leukocytes, it was interesting to discover whether factors in pancreatic homogenates could activate quiescent donor neutrophils as measured in this test.

Since pancreatic neutrophil activating factors are also present in species other than rats, there is an opportunity to obtain sufficient quantities of crude extracts for subsequent purification of these factors. In addition to experiments on chemiluminescent neutrophils enhanced in response to pancreatic homogenates, it was also interesting to determine whether pancreatic homogenates have superoxide-inducing properties as well as other types of properties. Since the endothelium plays an important role in neutrophil activation and adhesion is a major role in superoxide production, the effect of pancreatic homogenate applied to endothelial cell culture superoxide production was studied.

Finally, measuring chemiluminescence by these factors of neutrophil activation measured by chemiluminescence in the presence and absence of plasma, and with other established neutrophil activators It was interesting to compare. The interaction between neutrophils and the plasma components of the blood is important and is often a factor that is overlooked in assessing the presence and severity of different etiologies. One method of measuring neutrophil activation involves the use of lucigenin, luminol, or other chemiluminescent compounds that amplify photons produced by neutrophil production of oxygen free radical intermediates and other reactive products. It depends on the chemiluminescent used. (Delong et al.
(1989) J Chromatogr 492: 319-343; Ginsburg et al. (1993) Inflammation 17
: 227-243).

A particularly interesting species produced in the circulation by activated neutrophils is the superoxide anion (O ₂ ⁻ ). Many methods are currently used to measure this oxidizing species and NB
Includes T, cytochrome C, luminol and phenol red tests. Lucigen enhanced chemiluminescence is another such method. Unlike luminol-producing chemiluminescent, a relatively non-specific marker of superoxide, hydrogen peroxide and myeloperoxidase, lucigenin reacts specifically with superoxide to emit light. Lucigenin (dimethyldiacridinium nitrate) reacts in a two-step reaction (eg, Faulkner et al. (1993) Free Radic Biol.
Med 15: 447-451), which primarily detects superoxide (eg, US Pat.
94,541). Lucigenin chemiluminescence can be extinguished by superoxide-specific superoxide dismutase (SOD), which otherwise serves for the specific measurement of membrane-bound NADPH oxidase producing superoxide.

Although chemiluminescent tests are often performed on isolated leukocytes or other cells in an inflammatory state, a high percentage of activated neutrophils always adheres to blood vessels, and therefore neutrophils recovered from sample blood The sphere may not accurately reflect the actual degree of activation. Conversely, there are non-cellular components where many inflammatory mediators circulate in the blood, and thus are probably more reliable indicators of health. It is possible that plasma collected during an inflammatory condition provides a more accurate assessment of clinical severity than the isolated neutrophils themselves. Plasma recovery is an easier step than neutrophil isolation and may avoid potential artifacts more than can occur with neutrophil handling and isolation. Because autologous plasma has been shown to enhance the neutrophil-induced chemiluminescent response in vitro (Theron et al. (1994) Inflammation 18: 459-56
7) It was also interesting to determine whether the collected plasma served as a valuable substitute for isolated neutrophils in the measurement of superoxide chemiluminescence.

The lucigenin producing chemiluminescent as a means of measuring plasma concentrations was tested. Plasma measurements have advantages over isolated cells (eg, neutrophils) because they are a two-step method (centrifugation and measurement) and are subject to a large number of measurements and automation. The chemiluminescence produced by isolated human neutrophils in the presence and absence of standard activators (fMLP and platelet activating factor) at various concentrations of autologous plasma was compared.
In addition, homogenate activators from rat and pig pancreas were tested to gain a considerable understanding of their transient chemiluminescent activation properties in comparison to known activators, with rat and pig homogenates having low molecular weights (<3 kD ) Ultracentrifuged to separate fractions and measured separately for ability to activate neutrophils.

6.2 Methods 6.2.a Detailed Description Human blood was collected in heparinized Vacutainer tubes (10 ml) and 10
Centrifuged for minutes. Plasma was collected and 3 ml of lucigenin (N, N'-dimethyl-9,9'-bisacridinium dinitrate) (Sigma Chemical Co., St. Louis, MO) (1 mM storage in 1 ml of deionized water) The solution, 200 μM final concentration, near optimal concentration tested in Ohoi, et al. (26) was mixed in a small petri dish (60 mm diameter) for each measurement. In the latter variant of the test, a 25 mm diameter polyurethane disc is placed in a Petri dish to reduce the vessel diameter, followed by 1 ml of plasma mixed with 0.75 ml of lucigenin (1 mM stock solution) and only 100 μl of activator. Use less reagents. This small-scale version has only a small loss of signal,
When the activator volume and concentration are small (such as rat plasma collected before the shock protocol) or when a large number of measurements require autologous donors and it is desirable to apply the same plasma for each measurement. used. Normally collected from healthy volunteers into six Vacutainer tubes. Approximately eight measurements with this capacity in the original configuration
(3 ml plasma / measurement) and up to 25 measurements (1 ml / measurement) on the modified system.
When measuring whole blood samples, 3 ml of whole blood was replaced with 3 ml of plasma.

6.2.b Neutrophil Isolation A standard neutrophil isolation procedure was performed as described in Example 2, or in the pseudopodia formation test. Human blood (about 60 ml) from healthy volunteers was collected in heparinized Vacutainer tubes and transferred to a 60 ml syringe where it was sedimented on ice for 40-60 minutes. EDTA
It is important to use heparin rather than (ethylamineamine tetraacetic acid) as an anticoagulant because the calcium-chelating properties of EDTA can suppress neutrophil activation. The neutrophil-enriched plasma layer was collected and 3.5 ml Histopaque (Sigma Diagnostics, 17 × 100 mm, Falcon, Shreqsbury, Mass.) In a 12 ml polypropylene centrifuge tube.
(St. Louis, MO) and centrifuged at 600 G for 20 minutes. The precipitated neutrophils and red blood cells were then gently resuspended in 2 ml PBS (phosphate buffered solution). Resuspend cells carefully in 2.5 ml of 55% isotonic Percoll solution (Sigma Diagnostics) in deionized water.
cs, St. Louis, MO) and 2.5 ml of a 74% isotonic Percoll solution. The suspension was centrifuged for 15 minutes at 600G to, except the middle of the granules decomposition layer (granulyte layer), to achieve concentrations of suspended in 10 ⁶ neutrophils / ml in PBS.

For standard plasma measurements (eg, low molecular weight fraction and whole pancreatic homogenate), human blood (about 60 ml) from healthy volunteers was collected in heparinized Vacutainer tubes and centrifuged at 500 g for 10 minutes. . Carefully examine the plasma layer, including the buffy coat,
Decant using a sterile transfer pipette. The fraction was then warmed to room temperature and plasma measurements were taken. The plasma was diluted with sterile saline to achieve a plasma neutrophil concentration of 120 × 10 ³ neutrophils / ml.

For experiments with different plasma concentrations versus neutrophil activator, measurements were taken on isolated neutrophils,
Performed using isolated neutrophils in 10% and 30% autologous plasma, and aneutrophil plasma (confirmed by filtration with 1 μm filter and cell count).

6.2.c Activator Precipitate cell-free plasma and filter it with a 1 μm filter (Whatman 6780-2510, Swedesboro, N.
Obtained by filtration through J). The filtered plasma was confirmed visually by placing an aliquot on a microscope slide and testing in detail at 400 ×. For testing with different proportions of spiked plasma, a total of 5 ml was mixed in 1 ml lucigenin (1 mM), 1 ml activator, and 3 ml suspended neutrophils, PBS required for 10% or 30% total plasma concentration. It was made to contain either 2 ml suspended neutrophils + 1 ml cell-free plasma or 3 ml cell-free plasma. Activator (1 ml) was added to the mixture through the injection hole at t = 0 seconds. Activators used were pancreatic homogenate, total and low MW
Fraction, the chemotactic peptide N-formyl-methionyl-L-leucyl-
L-phenylalanine (fMLP) (10 ⁻⁶ M) (Sigma Chemical Co., St. Louis,
MO) and platelet activating factor (PAF) (10 ^-6 M) (Sigma Chemical Co., St. Loui)
s, MO). 1 ml PBS acted as a control activator.
Bovine erythrocyte-derived superoxide dismutase was purchased from Sigma Chemical Co., St.
Obtained from Louis, MO.

A rat pancreas homogenate was prepared as described above. Easy, male Wister rat,
Collect pancreas from 250-250 g, 3 months old, rinse with cold .25M sucrose solution, remove fat and excess tissue, weigh, 15 min, 1: 3 w / v ratio in Krebs-Henseleit solution using homogenizer And mixed. The mixture was then further diluted with a Krebs-Henseleit solution at a 1: 2 volume homogenate / volume ratio,
Incubate for 1.5 hours at 38 ° C. with shaking every 15 minutes. The eluate was filtered using a fixed rotor Amicon filter (Model S-30, Cent
(Ricon, Millipore Filter, Co., Beverly, MA). The ultrafiltration was kept at 4 ° C. until used.

For recovery of porcine pancreatic homogenate, male Hampshire pigs, 3-4 months old, 18-20 kg were used. Animals were prohibited from feeding 24 hours prior to surgery. Surgical anesthesia was induced with catamine (33 mg / kg / IM), atropine (0.05 mg / kg / IM) and sodium thiopental (10 mg / kg / IV) and maintained with a 1-2% halothane and oxygen combination. . Killing the animals with pentobarbital (120 mg / kg / IV)
The pancreas was immediately recovered and rinsed with cold saline. All pig experiments were performed at the University of Ca
This was done by Dan McKirnan at the UCSD Elliot Field Station, in accordance with the rules and requirements of the San Diego Animal Subjects Committee, California.

The pancreas was transferred to a 1: 1 saline: 0.25 M sucrose solution on ice and stored. Remove pancreatic fat and excess tissue, weigh 5 minutes, 1: 4 w /
Mix for 5 minutes in a commercial blender using the v ratio. The mixed homogenate was shaken vigorously and incubated for 2.5 hours at 38 ° C. with shaking every 15 minutes. The incubated homogenate was centrifuged at 800 G for 30 minutes, and the supernatant was passed through a 0.78 μm vacuum filter (Millipore Filter Co., Beberly, Mass.). The filtered eluate is then filtered at 3000 MW with a fixed rotor Amicon filter (Model S-30,
Centricon, Millipore Filter, Co., Beverly, MA). The ultrafiltration was kept at 4 ° C. until used. MALDI mass spectrometry of the selected sample confirmed no detectable signal above 3,000 MW. In fact, no signal was detected above 1,000 MW.

6.2.d Measurement The photons emitted from the resulting produced chemiluminescent were measured with a photomultiplier for more than 120 minutes (1 second light accumulation time) (Stanford Research 4000, S
unnyvale, CA), placed in a light-sealed device and a PC computer (486 Dell Computer).
Corp., Austin, TX) (SR467 Data Acquisiton Software Package, St.
anford Research Systems, Inc., Sunnyvale, CA). Photon counters and systems are available from the Department of Bioengineering, University of Californ.
ia, obtained from Dr. Richard Suzuki of San Diego, with slight modification of the experimental method.

Chemiluminescent experiments can be performed either continuously when a full time history is required (such as an assay curve with sharp spikes in amplitude), or batchwise when several samples are alternated (by hand) during the experiment. Made in mode. Batch methods have traditionally been used to avoid the potential for degradation of plasma and other biologicals that can occur if experiments were performed continuously. In batch mode, up to 12 samples can be simultaneously
Measured at minute intervals. For experiments that did not depend much on the passage of time, measurements were taken every 10 minutes.

6.2.e Endothelial Cell Nutrition Assay Endothelial cell chemiluminescent experiments in response to pancreatic homogenate were performed using confluent bovine arterial endothelial cells (BAEC) (Department of Bioengine).
ering, University of California, San Diego). BAECs were grown in a 60 mm diameter Petri dish at 37 ° C. in a controlled cell culture environment and standard RPMI medium (Gibco, Grand
Island, NY). For chemiluminescent measurements, cell cultures were checked for confluency by light microscopy to eliminate the potential for optical effects of the media remaining after rinsing with two standard Krebs Henseleit buffers. 1 ml Krebs-
Henserite buffer was added to the culture followed by 1 m 10 ^-3 lucigenin. For pancreatic homogenous experiments, 1 ml of whole pancreatic homogenate or 1 ml of low molecular weight pancreatic homogenate was added. One ml of Krebs-Henseleit solution was added to the control culture. Chemiluminescence was measured using light accumulation time of 1 minute as described above. All endothelial chemiluminescent tests were performed in duplicate.

6.2.f Chemiluminescent System Calibrator The calibration of the lucigenin chemiluminescent assay was performed by adding lucigenin to 3 ml buffered saline solution. A known concentration of potassium superoxide (KO ₂ ) (Sigm
a Chemicals, St. Louis, MO) was added and the chemiluminescence was measured. A linear response was obtained between 1 nM and 10 μM. The potassium superoxide curve is K
Although O ₂ reacts spontaneously to form superoxide in a 1: 1 ratio (allowing for direct quantification of the absolute concentration of superoxide), it is preferred for superoxide calibration, but xanthine oxidase has various levels. create a super oxidase and H ₂ O _2, depending on the experimental conditions.

As described above (Example 3), xanthine (and hypoxanthine) was
Reacts with oxidase to produce superoxide and hydrogen peroxide. Under static conditions
, Xanthine oxidase exists as xanthine dehydrogenase, and NAD ⁺ To make NADH and uric acid.

Included as a general indicator of the relative amount of free radicals produced and to facilitate comparison with the results of other groups. Hydrogen peroxide does not react with lucigenin and no signal is obtained (curves not shown). DMSO (dimethyl sulfoxide) should not be used as a reagent because it reacts strongly in plasma to produce chemiluminescent.

The results are expressed as mean ± SD, except for continuous chemiluminescent measurements where standard deviations have been omitted for readability. Student's t-test (two-tailed unpaired) was used for all comparisons. Differences with p <0.05 were considered significant.

6.3 Results For the representative time course of control plasma without the addition of activator, the average maximum (stable state) value for each experiment (n = 100 +) was about 3300 ± 500 at about 40 seconds.
Counts / sec. The time course is characteristic of lucigenin-measuring chemiluminescence and appears to be related to the interaction between neutrophils and lucigenin.

The results of the chemiluminescent test of low molecular weight (n = 6) and whole rat pancreatic homogenate (n = 6) were significant (P <0.05) compared to control (n = 3) in the 40 min experiment. From P <0.001). Plasma chemiluminescent (PMN concentration 150 × 103 ml) of whole rat pancreatic homogenate (n = 6) and low molecular weight (<3 kD) rat pancreatic homogenate (n = 6) compared to control (n = 3). Control and activated samples show a typical increase in chemiluminescence over the time course of the experiment. It is not clear whether this increase is due to a filling phenomenon or possibly due to the diffusion of lucigenin in otherwise well-mixed samples. The results for pancreatic homogenate added to cells alone are consistent with this. The results of the plasma experiments obtained after the addition of rat pancreatic homogenate (1 ml of supernatant solution prepared as described above, approximately 11 μg total protein / μl) show that even isolated neutrophils have enormous amounts of superoxide-producing chemiluminescence. It differs significantly from the previous curve in that there is a significant increase.

The application of low molecular weight (n = 5) and whole porcine pancreatic homogenate (n = 5) to plasma also showed significant chemiluminescence (P <0.05-P) compared to control (n = 5).
<0.005). Results were performed for neutrophils alone (in PBS), neutrophils in 10% analagous plasma, neutrophils in 30% analagous plasma, and plasma alone. Unstimulated neutrophils alone or plasma without cells also did not produce visible levels of chemiluminescence when taken from healthy donors. this is,
There may be a change in the inflammatory condition with circulating superoxide donors (such as circulating xanthine oxidase) as well as the resulting activated neutrophils that may be present in a sample of plasma without cells.

Experiments with human neutrophils alone after stimulation with peptide activator fMLP (1 ml of 10 ⁻⁶ M), neutrophils in 10% autologous plasma, neutrophils in autologous 30% and plasma alone The results show that even somewhat higher concentrations of this drug cannot essentially activate NADPH oxidase. At increased levels of plasma concentration, there was a concomitant increase in superoxide production. With plasma alone, superoxide production is very low, as would be expected with fMLP application to cell-free media. Again, in inflammatory conditions where there is a visible amount of circulating activator, this may not be true.

In similar experiments after addition of platelet activating factor (1 ml of 10 ⁻⁶ M), these curves show a general trend according to the other receptor-mediated activator, fMLP, but at slightly lower levels. Is shown.

The results of plasma experiments after addition of whole fraction rat pancreatic homogenate (1 ml μg protein / μl) show that these curves show that even isolated neutrophils and superoxide producing chemiluminescent at different levels of plasma It shows a significant difference from the previous curve in that there is a huge increase. There is also a slight increase in chemiluminescence from plasma-only samples incubated with pancreatic homogenate.

Incubation of rat pancreatic homogenate, low molecular weight and whole homogenate results in a dramatic increase in chemiluminescence when added to confluent bovine arterial endothelial cell (BAEC) culture. Control BAEC cultures showed a verifiable increase in chemiluminescence without delay, which was attributed to the temperature sensitivity of the photomultiplier tubes, which showed a slight increase in basal photon count with instrument warming. This increase is less than 1% of the normal measurement when measured with a 1 second light storage time, but is noticeable when a lower concentration measurement of endothelial producing chemiluminescent is measured with 1 spectrometer storage time. is there. Therefore, in all endothelial cell culture experiments, this temperature was precisely controlled by the length of the warming time of the machine. The result of the control experiment was 96
The average differed by 2 ± 187 photons / min (about 3-5% of the total control sample photon count).

6.4 Discussion Quantification of neutrophil activation can be done in many different ways. These are methods such as NBT, pseudopodia formation, chemiluminescence and the like, each of which measures a specific parameter for a cellular response to a stimulus. For example, pseudopodia formation measures actin polymerization that occurs when neutrophils respond to some chemotactic activator. Other responses to neutrophil activation include formation of oxygen free radicals and degranulation of primary and secondary granules following promotion of the NADPH oxidase system. These are all activated neutrophil responses but need not be coupled. Different activators activate various components of the neutrophil cytoplasm and membrane. Peroxide-induced chemiluminescence is a "solid" indicator of neutrophil activation and does not require operator judgment (compared to NBT and pseudopodia formation), so that pseudopodia formation and NBT It was investigated whether the pancreatic equivalents that activate neutrophils, as seen in the tests, are mediated by luminigenin chemiluminescence.

According to this study, incubation equivalents from rat and porcine pancreas activate neutrophils. This is measured by the in vitro peroxide chemiluminescence formation. No other tissue equivalents were activated in vitro. Rat and pig homogeneity is produced in a similar manner and has approximately the same strength. In addition, the pig homogeneity is not so diluted. These results indicate that there may be common factors that are not species independent. In rats and pigs, there is considerable chemiluminescence in the whole pancreatic equivalent and small molecule fraction. Whole pancreas homogeneity contains a low molecular fraction but was assumed as follows. Any neutrophil activator from the pancreas is originally a protease and has a molecular weight of about 30 KD to 100 KD (see Example 5). The fact that the small molecule (3 <KD) component contained in rats and pigs activates the production of NADPH oxidase in neutrophils does not rule out this view. Larger molecular weight proteases regulate neutrophil responses to other activators and are probably synergistic in these responses. Small molecule activators, although not expected, indicate the presence of small peptidomimetic or lipidic substances in the pancreas where mau activates neutrophils. The relative strength of the small molecule activator is at least as high as the total molecular weight fraction,
It has been shown that the small molecule fraction is of primary importance for the activation of PH oxidase producing peroxide. This has some differences from the data on pseudopodia formation. According to pseudopodia formation, whole pancreatic equivalents are consistently slightly more potent than small molecule fractions in promoting actin polymerization. Note again that the process is not coupled.

In addition to peroxide-induced chemiluminescence by pancreatic equivalents to neutrophils, pancreatic equivalents also activate endothelium in vitro. This is assayed with lutigenin chemiluminescence. The relative difference between the strength of the whole pancreatic equivalent (very strongly activated over 1 hour) and the small molecule fraction (very weakly activated during that time) was due to the neutrophil chemiluminescence study. Very different from the differences seen. Although neutrophil-activated chemiluminescence appears to be equally present in all and low molecular fractions, only high molecular weight endothelial activators are present in the results of endothelial activation experiments. In each case, the presence of high and low molecular weight activators is not hindered. A possible source of high molecular weight endothelial activators is pancreatic (serine) protease, which is discussed in detail in Example 7.

It is interesting to observe the relative strengths of the various activators required to promote the NADPH oxidase system at various concentrations. FML added to isolated neutrophils
P is not particularly active in peroxide-induced chemiluminescence production. PAK, another receptor-mediated neutrophil activator, is similarly inactive in suspended neutrophils. The addition of varying amounts of plasma to neutrophil cells greatly increases peroxide-induced chemiluminescence. In conclusion, it can be said that neutrophils require appropriate extracellular ATP to cause a respiratory burst.

The results of the chemiluminescent response of pancreatic equivalents are particularly surprising. Equivalents have no intrinsic chemiluminescence stimulatory properties, but adding equality to cell-free plasma results in a slight increase in chemiluminescence. This was not seen with other activators. A further noteworthy result is that only pancreatic equivalents added to suspended neutrophils cause a dramatic and transient increase in peroxide-induced chemiluminescence. Evidently, the homogeneity at the concentrations used is a very potent activator of human neutrophils in vitro. Presumably, some ATP-producing substances are present in pancreatic equivalents and behave the same in autologous plasma. Or,
Differences in the NADPH oxidase activation pathway may be involved. For example, when the non-receptor-dependent activator phorbol ester PMA activates peroxide-dependent chemiluminescence in the absence of plasma. in this way,
Equivalence is very useful in assays to screen for inhibitors of its activation.

Combining small molecule stimuli with high molecular weight priming agents (such as serine proteases that can directly cleave CD41 ligand) can reduce the need for the addition of plasma. The addition of 10% plasma greatly enhances the response of isolated neutrophils to pancreatic equivalents.
The magnitude of chemiluminescence derived from isolated neutrophils is an order of magnitude greater than any activation produced by fMLP or PAF by mixing with 10% plasma and activating in pancreatic equivalents. The time course of chemiluminescence also appears to be slowed by the addition of plasma, ie, as the plasma is increased, to reduce peroxide-dependent chemiluminescence. Possible causes of this decrease in chemiluminescence include a high rate of chemiluminescence due to the predominant antiprotease screen and the equivalent protease fraction present in healthy plasma. It is clear from the relative magnitude of total and low molecular weight peroxide-induced chemiluminescence that protease and antiprotease interactions cannot be the only result of increased plasma concentrations. In isolated chemiluminescence experiments, addition of high concentrations of the major pancreatic proteases trypsin (1000 U / ml) and chymotrypsin to plasma (neutrophil concentration 155 × 10 ³ neutrophils / ml plasma) resulted in almost no activation. It does not occur.

The end result of these chemiluminescence measurements is to establish a possible method for assaying peroxide production in vitro. As mentioned above, most existing lutigenin chemiluminescence methods use isolated neutrophils from patients. The chemiluminescence obtained by stimulation with a known stimulus such as PMA is then measured. This type of method has several disadvantages. Among other things, it requires isolated neutrophils, which is time consuming. Requires additional reagents. Further problem is that the activation of neutrophils is different for some stimuli, which may vary in intensity (neutrophils are centrifuged at least twice and sedimented in dextran Resulting in various osmolarities). In addition, as mentioned above, neutrophils in inflamed patients are already activated, and sampling venous blood from such patients does not always give an accurate value for the percentage of activated blood. Activated neutrophils tend to adhere to the endothelium in microcirculation and are unlikely to recover in venous blood.

The method used in this study ameliorates the above-mentioned problems. That is, it is simple, fast, reproducible, and inexpensive. This method can be used in the usual way. That is, fresh patient blood is centrifuged and peroxide formation in plasma is measured. Often, control plasma from healthy individuals, as well as plasma from other patients, are used as carriers to test activation of different substances. Other patient blood methods provide neutrophils in autologous plasma, eliminating the need for large amounts of patient blood. The ability to activate resting neutrophils can be measured with as little as 100 μl of plasma (and even smaller using a new small amount of conformation). Accurate results with this method can be obtained in as little as 1 hour (10 minutes centrifugation, 10 minutes instrument, 40 minutes measurement). Because the number of neutrophils in centrifuged plasma is much lower than isolated neutrophils in autologous plasma, the relative levels of chemiluminescence are diminished. In normal (control) plasma, the maximal response at all large values (> 100 experiments with 5 or more donors) is 20-50
It is 1500-6000 counts / second per minute. The normal range is 3000+ in about 40 minutes
/ -500 number / second. This value depends on the donor's illness, antibiotics and, more interestingly, the consumption of fatty foods.

The small molecule and total fraction of pancreatic equivalents significantly increased the chemiluminescence of peroxide production from donor neutrophils and plasma compared to control values. In addition, whole pancreatic equivalents significantly increased peroxide production by BAEC endothelial cell cultures. The chemiluminescent activation produced by neutrophils and plasma incubated with pancreatic equivalents (9: 1 vol / wt) was significantly greater than that expressed with corresponding amounts of known activators fMLP and PAF. From this,
It is believed that potent factors, such as neutrophils, that can activate cardiovascular cells are present in the pancreas.

Example 7 Involvement of Proteases in Pancreatic Neutrophil Activators Summary The level of neutrophil activation is increased by visceral artery occlusion (SAO) shock. The measurement is made on pseudopodia formation in donor neutrophils exposed to shock activation. Equivalents from rat abdominal organs do not significantly activate isolated naive neutrophils except pancreatic equivalents. Pancreatic equivalents contain factors that highly activate neutrophils in vitro. Because of the high levels of proteases in this organ, the mechanism of neutrophil activation may be related to proteases. The reported effects of protease inhibitors on shock, the deleterious systemic effects of circulating proteases, and neutrophil activation by various known proteases allow for a direct mechanism of neutrophil activation by pancreatic proteases There is.

To study this, the ability of pancreatic equivalents to activate human isolated naive neutrophils was performed in the presence or absence of various protease inhibitors. Randomly selected rats were weighed, anesthetized, and arterial and venous catheterized. Laparotomy and phlebotomy were performed. The pancreas was removed immediately, placed in 0.25 M sucrose solution and homogenized in 1: 9 (w / v) Krebs-Henseleit solution. Equal aliquots were mixed with various protease inhibitors. Serine protease inhibitors were effective and inhibited the activation of human neutrophils incubated with rat pancreatic equivalents. The protease inhibitor with the greatest in vitro potency was futan (nafamostat mesilate), which abolished pancreatic equivalent-induced activation.

[0324] Since pancreatic equivalents activate endothelial cell cultures and naive neutrophils in vitro, it was tested to see if pancreatic equivalents activate other tissue equivalents. In vitro neutrophil activation by pancreatic equivalents was inhibited by the addition of a serine protease inhibitor. It is therefore of interest whether neutrophil activation by non-pancreatic tissue occurs when pancreatic equivalents or exogenous serine proteases are added to other organs. Rat organs collected and homogenized in addition to the pancreas are spleen, proximal small intestine, heart and liver. Aliquots of each sample were mixed with unstimulated amounts of pancreatic equivalents or serine protease trypsin, chymotrypsin, or both. The suspension was incubated for 2.5 hours at 38 ° C. and neutrophil actin polymerization (pseudopodia formation) was measured. Neutrophil activation, which resulted in a significant increase (p <0.01), was due to substimulatory amounts of pancreatic equivalents or tissue equivalents incubated with serine protease. Activation was not increased when equivalents from organs other than the pancreas were used. These results show that tissue equivalents incubated with serine proteases contain factors that activate neutrophils in vitro. The pancreas may serve as an endogenous source for neutrophil activation.

7.1 Introduction The splanchnic artery occlusion (SAO) shock releases circulating factors into the blood that have the ability to activate neutrophils in vitro, in addition to other etiologies such as bleeding and endotoxin shock. (See, Example 3). Tissue equivalents from the pancreas, unlike those from other organs examined, activate naive neutrophils. This is detected in the actin polymerization and peroxide formation tests (see, Example 5). The pancreas is also considered an endogenous source of neutrophil activators in vivo. Such factors are released in shock and other etiologies (such as malnutrition and sepsis) and may contribute to initial neutrophil activation and priming.

In this study, the identification of a product in the pancreas that leads to neutrophil activation in vitro was performed. The pancreas is an essential constituent in the visceral area and has two distinct digestive functions,
Plays a major role in endocrine and exocrine processes. These two functions make use of two different cells in the pancreas. The β cells of the islets of Langerhans exercise the endocrine function of the pancreas and produce insulin directly in the bloodstream in response to increasing blood sugar levels.
Another cell in the pancreas controls endocrine functions in the body. Acinar cells hold almost inactive proenzymes and other latent phenolic substances. These pancreatic substances are released in response to the digestive process of the digestive tract. The major ones are proteolytic enzymes, which are released from the inactive zymogen form to the active enzyme by the action of trypsin and are themselves cleaved from the inactive zymogen by the intestinal enzyme enteropeptidase (Table 7.1: Rinderknecht (1993); Chapter 12, The Pancreas:
Biology, Pathology, and Disease, Go et al. , Ed, Raven Pres
s, NY, pp 219-251) Other pancreatic enzymes include lipase, carboxylester hydrolase, amylase, ribonuclease, and deoxyribonuclease I.

[Table 2]

In the normal functioning pancreas, activation of potential autocatalytic enzymes is controlled by limiting trypsin activation until transport of trypsin to the gastrointestinal tract. In addition, pancreatic enzyme antiproteases such as pancreatic secretory trypsin inhibitor (PSTI) coexist. It is present in an amount sufficient to inactivate pancreatic trypsin by up to 20%. PSTI limits the proteolytic activity that can result from inappropriate protease (self) activation. When released into plasma, pancreatic proteases are inactivated by protease inhibitors such as α ₁ -proteinase inhibitor (α ₁ -antitrypsin), α ₂ -macroglobulin, inter α ₁ -trypsin inhibitor, α ₁ -antichymotrypsin Can be Of these inhibitors, α ₁ -proteinase inhibition has the highest concentration, about 90% of the plasma protease screen. This antiprotease "screen" inactivates all proteases that pass through the circulation. Despite a well-controlled release mechanism for pancreatic digestive enzymes, these pancreatic proteases are released in various pathological conditions and play important roles in various pathological conditions such as pancreatitis and shock There is. With depletion of the antiprotease screen, pancreatic and neutrophil proteases circulate freely and cause tissue destruction of the entire system. Pancreatic derived proteases are thought to play a role in initiating endothelial free radical production. this is,
By conversion of membrane-bound xanthine, dehydrogenase to xanthine oxidase. In reperfusion after ischemia, membrane-bound and circulating xanthine oxidase produces large amounts of oxygen free radicals, causing tissue damage and cytokine activation. Thus, improper release of pancreatic enzymes initiates neutrophil activation as seen in shock and pancreatitis.

In studies by measuring the action of protease inhibitors, it was investigated whether the in vitro neutrophil activator from pancreas was originally a protease. Of particular interest is the measurement of pancreatic serine protease activity, which includes most of the etiological effects attributable to proteases. Given the serine moieties of the catalytic amino acids triad His-57, Asp-102, and Ser-195 that create active proteolytic sites, proteases from this family are inhibited to varying degrees by serine proteases. This inhibition depends on the conformation of the particular protease involved. Assays for different and diverse protease inhibitors were performed for their ability to inhibit neutrophil actin polymerization (pseudopodia formation) by rat pancreatic equivalent in vitro application.

Another interest is whether the inhibitory effect of the protease on neutrophil activation is homogenous or neutrophil-dependent. To answer this question, we performed a series of experiments. Neutrophils were incubated with protease inhibitors and then all unbound protease inhibitors were washed out. The inhibition seen in the equivalent response to the "washed" sample was compared to neutrophils that had not been washed out and were incubated with protease inhibitors still present in the buffer. In this way, it would be possible to determine whether protease inhibition is associated with pancreatic equivalents or neutrophil counts themselves.

It has been hypothesized that pancreatic proteases play a similar role in other organs, in addition to degeneration of tissues in the pancreas and possible formation of neutrophil activators. Then, the ability of the pancreatic equivalent and its major proteases trypsin and chymotrypsin to induce other tissues and express neutrophil activators was studied.

7.2 Methods Rat equivalents were collected. Details are described in Example 5. Briefly, male Wistar rats (250-350 g) were placed in a controlled environment and fed a standard pellet diet for at least 3 days after the start of the experiment. The artery and vein of the thigh were cannulated, and general anesthesia was performed with pentobarbital (50 mg / kg im). Phlebotomy, heart, liver,
The spleen, small intestine and pancreas were removed. The organs were washed immediately and washed with cold 0.25M sucrose solution. These organs were vigorously homogenized with Krebs-Henseleit solution (1: 9 w / v). Aliquots were centrifuged at 500 G for 10 minutes. The pancreatic equivalents were incubated for 2.5 hours at 28 ° C. and agitated multiple times. For the incubated pancreatic equivalents and controls, human donor naive neutrophils were tested for pseudopodia formation as described in Example 2.

The pseudopodia formation test was repeated to determine whether neutrophil activation by pancreatic equivalents was protease-dependent. Isolated neutrophils were first incubated with various protease inhibitors (50 μl) for 10 minutes, then pancreatic equivalents (5
0 μl) was added and the mixture was further incubated for 10 minutes. Inhibitors used were phenylmethylsulfonyl fluoride (PMSF) (1 mM), Couplete ™ with and without EDTA (1 tablet / 20 ml), benzamidine (1 150 μM), futan (Nafamostat Mesilate) (0.1 mg). / Ml) and aprotinin (20 μM). All protease inhibitors were
Gifted by Dr. Tony E, Hugl of the cripps Clinic and Research Institute. Only Couplete ™, an all-purpose protease inhibitor, was purchased from Boehringer Mannheim, Indianapolis, IN. One of the components of Couplete, EDTA (ethylenediaminetetraacetic acid), is a standard anticoagulant and a divalent cation chelate. Since the calcium scavenging effect of EDTA also inhibits the neutrophil response to the stimulus, the inhibitory effect of Couplet was assayed with or without the addition of 70 μM MgCl ₂ and bound to soluble EDTA as indicated by the company.

A new series of neutrophil pseudopodia experiments was performed to determine whether protease inhibitors act on pancreatic equivalents or on neutrophils themselves. Three groups were used. In the control group, isolated human neutrophils (100 μl of 10 ⁶ cells / ml) were washed twice with D-PBS and incubated with 50 μl of pancreatic equivalent for 10 minutes. In the washed group, isolated human neutrophils (100 µl of 10 ⁶ cells / ml) were incubated with 50 µl of phthalate (0.1 mg / ml) for 10 minutes, washed twice with D-PBS, and unbound phthalate was removed. Removed and then incubated with 50 μl of pancreatic equivalent for 10 minutes. In the inhibitor group, the isolated human neutrophils (100 μl of 10 ⁶ cells / ml) were washed twice with D-PBS and 50 μl phthalane (0.1 mg / ml)
And 50 μl of pancreatic equivalent. This combination had been previously incubated together for 10 minutes. Appropriate controls were recorded along with positive controls (10 ^-7 M fMLP) to determine the ability of washed cells to respond to stimuli. Formyl-methionyl-leucyl-phenylalanine (fMLP
) Is from Sigma Chemical Company, St. Louis, MO. Purchased from.

To investigate the additional in vitro effects of serine proteases on other organs,
The spleen, heart, small intestine, and liver equivalents were divided into aliquots and incubated with either: Sub-activating concentrations of pancreatic equivalents (100 μl filtered pancreatic equivalents / 3 ml organ equivalents) (confirmed in previous assay), trypsin (1300 U
/ Ml equivalent), chymotrypsin (104U / ml equivalent), trypsin (13
(00 U / ml) + chymotrypsin (52 U / ml) or a corresponding amount of a control solution (Krebs-Henseleit solution). These unincubated samples were incubated, usually at 38 ° C. for 2.5 hours with occasional agitation. Once prepared, the samples were measured within 24 hours to give a half-life in the range of about 24-48 hours with reduced activation at the test equivalent.

In each separate experiment, the ability of trypsin and chymotrypsin, as well as their precursors trypsinogen and chymotrypsinogen, to activate naive neutrophils was examined by pseudopodia formation. The ability of trypsinogen and chymotrypsinogen activated by trypsin to activate resting neutrophils was also examined. Trypsin (porcine pancreatic type 11-S), α-chymotrypsin (bovine pancreatic type II), trypsinogen (bovine pancreatic type) and α-chymotrypsinogen (bovine pancreatic type II) are available from Sigma Chemical Company, St. Purchased from Louis, MO.

The results are expressed as mean ± SD for all samples. Student's pair
Using the assay, pseudopodia formation was examined on samples with and without activator addition. The 2-til Student's test was used for all other comparisons. Differences with p <0.05 were considered significant.

7.3 Results The reduction in neutrophil activation by protease inhibitors on neutrophil pseudopodia formation by rat pancreatic equivalents was different for the protease inhibitors used.
Benzamidine (n = 7) and aprotin (n = 9) had minimal but significant results. That is, the substantial inhibition of neutrophil activation by pancreatic equivalents is 85%
. From 1 ± 11.5% to 41.5 ± 24.6% and 51 ± 17.2% respectively
(P, 0.05). Couplete was effective in reducing response to pancreatic equivalents (46.8 ± 13.4%) (p <0.0005). Inhibition of neutrophil pseudopodia activation was determined by PMSF (n = 5) (40.8 ± 12.9%) and futan (n
= 5) (30.3 ± 11.8%) (p <0.001). In subsequent experiments, only futan was used as an inhibitor of neutrophil activation by pancreatic equivalents. Inhibition of neutrophil activation by protease inhibitors was dose-dependent in pseudopodia formation assays. Application of protease inhibition was not inflammatory at the concentrations used.

Experiments on the mechanism by which protease inhibitors inhibit neutrophil activation by pancreatic equivalents were less clear. Isolated human neutrophils washed twice with D-PBS were not substantially activated (9% activated (n = 2)), but fM
LP (74% activation (n = 2)) and pancreatic equivalents in control group (69.8 ± 21)
% Activation (n = 10)). Inhibition of activation by protease inhibitors (0.1 mg / ml) was slight.
Neutrophils were first incubated with futan and washed twice to remove excess futan (washed group (53.5 ± 31% activated (n = 8)), neutrophils were washed with futan and after washing the pancreas Group incubated with equality (inhibitor group) (51
. 5 ± 34% activation (n = 8)). Pseudopodia tests of pancreatic equivalent were compared for naive washed (2 ×) neutrophils (control group [n = 10]), futanized and washed (2 ×) neutrophils (washed group [n = 8). ]), A group in which na ー ブ ve washed (2 ×) neutrophils were incubated with ethane and then with pancreatic equivalents (inhibitor group [n = 8]
). Untreated naive cells (washed 2x) and fMLP treated (10-7M)
Neutrophils are negative and positive controls, respectively. Between the washing group and the inhibitor group,
And significant difference between control group and washed group or inhibitor group (p = 0.1)
Was not found.

Incubation of pancreatic equivalents with other organ equivalents at sub-activating concentrations for 2.5 hours at 38 ° C. resulted in a significant increase in pseudopodia formation rates. 8.3 with liver equivalent (n = 6)
± 5.2% to 38 ± 26.5% (p <0.05), 27 for intestinal equivalent (n = 6)
. It was 7 ± 19.9% to 78 ± 12.1% (p <0.001). There was no significant increase between 15 ± 14.1% to 35.3 ± 37.2% in pancreatic incubated hearts (n = 6) and 33.1 ± 15.8 in spleen equivalents (n = 6). % To 43.
It was 2 ± 23.2%. These are comparisons with non-pancreatic incubation equivalents (n = 5). There was also a significant difference in pseudopodia activation between pancreatic incubated controls and pancreatic incubated spleen equivalents (p <0.01) and intestinal equivalents (p <0.0
01) and a significant increase in pseudopodia formation was pancreatic incubated heart and liver homogeneity. Slightly significant were control activation (10.5 ± 2.5) and pancreas incubation control (18.1 ± 7.5%) (p = 0.05). (% Neutrophils representing pseudopodia; organ homogeneity (n = 5),
Or organ equivalents incubated with filtered pancreatic equivalents (n = 5) (100
after mixing with μl pancreatic equivalent / 3 ml organ equivalent).

The organ equivalent was increased by 2.5 with serine protease, trypsin + chymotrypsin.
When incubated at 38 ° C. for a time, there was a significant increase in% pseudopodia formation in all tissue equivalents (n = 6) as compared to the incubation equivalent without protease (n = 5). Increased activation increased from 8.3 ± 5.2% to 74.4 ± 24.
4%, 33.1 ± 15.8% to 72.75 ± 17.4% for spleen equivalents, 15 ± 14.1% to 78.9 ± 13.3% for cardiac equivalents, 27 for intestinal equivalents 0.7 ± 19.
It was 95.7% to 65.7 ± 8.9%. Protease incubation control (n =
There was also a significant difference between 4) and all equivalents (spleen equivalents p <0.01
, All other tissues p <0.001). There was no significant difference between control activation (10.5 ± 2.5%) and the protease incubation control (9.6 ± 5%) (
Percent of neutrophils representing pseudopodia. Organ equivalents (n = 5) or after mixing with serine protease, chymotrypsin (52 U / ml equivalent) and organ equivalents incubated with trypsin (1300 U / ml equivalent) (n = 6).

Incubation of organ equivalents with the serine protease trypsin alone at 38 ° C. for 2.5 hours resulted in pseudopodia formation in all tissue equivalents (n = 4) compared to the incubation equivalent without trypsin (n = 5). % Significant increase (p <0.
001). Increased liver equivalent activation increased from 8.3 ± 5.2% to 84.5 ± 9.47%
Increased spleen activation from 33.1 ± 15.8% to 67 ± 12.2%, increased cardiac activation from 15 ± 14.1% to 87.75 ± 7.1%, increased intestinal activation Is 27.7
It was ± 19.9% to 69.2 ± 12.8%. Significant differences were also observed between the trypsin-incubated control (n = 2) and all tissue equivalents (p <0.01 for intestinal equivalents, p <0.001 for all other tissues). Control activation (10.5 ± 2.5%) and trypsin incubation control (8.5 ± 0.8)
%). (% Neutrophils representing pseudopodia. Organ homogeneity (
n = 5) or after mixing with organ equivalents (n = 4) incubated with trypsin (2600 U / ml organ equivalents).

A single sample of organ equivalents was prepared with the serine protease chymotrypsin for 2.5 days.
Incubation at 38 ° C. for a time increased the proportion of pseudopodia formation in all tissue equivalents. The increase in% chymotrypsin-induced pseudopodia formation was greatest in liver and heart equivalents, from 7.4 ± 5.1% to 99% and 15 ± 13% to 9%, respectively.
5%. Increased spleen activation ranged from 29.1 ± 17.9% to 47%, and increased intestinal activation ranged from 24.3 ± 29.3% to 80%. The increase in chymotrypsin-incubated control sample activation was from 8.1 ± 5.1% to 42%, much greater than normally seen with this material (Author's Note) (% neutrophils representing pseudopodia) .
After mixing with either organ equivalents (n = 5) or organ equivalents (n = 1) incubated with chymotrypsin (104 U / ml organ equivalent).

7.4 Discussion These studies show that neutrophil activation by pancreatic equivalents is inhibited by serine protease inhibitors in vitro, as examined by actin polymerization. Of the protease inhibitors tested, futan was the most effective at inhibiting pancreatic neutrophil activator in vitro. Futan or FU
T-175, nafamostat mesilate (6-amidino-2-naphthyl, p-guanidinobenzoate dimethanesulfonate), is a known compound, a low molecular weight serine protease inhibitor, made by Torii & Company (Tokyo). A wide range of inhibitors, including trypsin, chymotrypsin, elastase, kallikrein, plasmin, thrombin, enterokinase, Cir and C
l esterase, inhibiting phospholipase A _2. The primary clinical use of futan in Japan is coagulation disorders such as seeded intravascular coagulation (DIC) and acute pancreatitis.

In vitro, futan inhibits dymosan-induced chemiluminescence activation by neutrophils, but has no free radical scavenger action. This effect has been criticized by other serine protease inhibitors. Futanes dose-dependently inhibit neutrophil actin polymerization by pancreatic equivalents of incubated rats.

Inhibition of neutrophil activation by rat pancreatic equivalents by futan indicates the involvement of serine proteases in the activation process. This involvement is as a soluble activator of neutrophils or as an endogenous component of the neutrophil surface receptor response. For this reason, futan can be used as a drug for hypoactivation therapy.

Since the incubated pancreatic equivalents contain significant levels of protease,
This enzyme is a candidate for a large molecular weight (20-40 KD) neutrophil activator. Serine proteases such as trypsin and chymotrypsin can amplify neutrophil activation against stimuli such as fMLP and phorbol myristate acetate (PMA) (amplification intensity: cathepsin G>chymotrypsin>elastase> trypsin). Apoptosis has been reported, but it has not been reported that these proteases activate neutrophils.

This has been demonstrated in this study. The use of trypsin and chymotrypsin does not result in neutrophil activation either by pseudopodia formation or chemiluminescence (see Example 6.4), but rather (organ-equivalent) neutrophil activation. A chymotrypsin-like protease on the surface of neutrophils appears to be involved. This enzyme is inhibited by protease inhibitors. This receptor molecule is thought to be CD43 and appears to act as a "functional barrier" for neutrophil activation. In addition,
This activation is tested with opsonized dimosan and PMA. Proteolytic (chymotrypsin-like) cleavage of CD43 may be a necessary event for neutrophil activation.

We determined whether the inhibition of neutrophil activation by pancreatic equivalents in vitro by serine protease inhibitors was dependent on neutrophil equivalent factor or protease inhibitor inactivation of neutrophils. . Neutrophils washed out of free (circulating) protease inhibitor phthalane showed comparable inhibition in pancreatic equivalent-induced activation as neutrophils incubated with futan plus pancreatic equivalent. The level of inhibition in this experiment was lower than in other futan inhibition studies (51-53% inhibition versus a typical inhibition of neutrophil activation of about 30%), so a clear conclusion can be drawn from this experiment. Did not. Significant neutrophil inhibition was achieved when protease inhibitors were used followed by washing out unbound inhibitors, or when protease inhibitors were used directly with neutrophils with equal quality. Therefore, the inhibitory action of futan
It is predominantly directed at the neutrophil membrane chymotrypsin-like receptor, but not by factors in the homogeneity itself. The lack of complete inhibition in the washed group may be due to the fact that the activator promotes neutrophils, lacking total receptor binding after washing. In the unwashed group and the inhibitor group, the phthalane binding of free pancreatic protease
Effective ethane concentrations can be reduced, inhibiting the ability of protease inhibitors to compete effectively at the binding site. These results suggest that pancreatic proteases probably play a synergistic role in 'priming' neutrophils for activation, but are not endogenous pancreatic neutrophil activators.

However, this finding does not relate to the role of pancreatic proteases and pancreatic neutrophil activators in synthesis. It is known that pancreatic enzymes are closely involved in the self-destruction of pancreatic tissue and the release of toxic factors. In addition to trypsin and chymotrypsin, other enzymes that may be important in the pathological pancreas include lipases and elastases. These enzymes are involved in the pancreatic autolysis process.

Incubation of initially inactive tissue with sub-activating concentrations of pancreatic equivalents yields significant levels of neutrophil activator in the organs tested (spleen, heart, liver, small intestine). This result is reproduced when the tissue is incubated with either trypsin or chymotrypsin, or both. Based on this finding,
For neutrophil activation in adverse conditions, such as shock, the pancreas has a distinct in vivo role, and circulating pancreatic enzymes can be routinely measured in disease states. Protease releasing neutrophil activators have not been clearly identified. One possible component factor is a platelet activating factor (PAF) -like substance. This substance
It has been reported that it is synthesized and released from endothelium in response to stimulation by proteases such as chymotrypsin and trypsin. It is also possible that the release of pancreatic proteases into the circulatory system may result in the formation and release of neutrophil activator by organs other than the pancreas, especially the compromising event of the anti-protease plasma screen.

It is possible that neutrophil activators released by protease incubation in tissues that are initially inactive are the same as those released by the pancreas itself. There is some evidence to the contrary. First, pancreatic homogenates increase potency to some extent after incubation at 38 ° C. for 2.5 hours, maximizing the proteolytic process, but still having neutrophil stimulating activity after incubation. This means that protease activation is not required for expression of this factor (
See Example 5). Second, the half-life of in vitro neutrophil activation potency differs significantly between pancreatic homogenates and protease-incubated tissues. The neutrophil activating component of the pancreatic homogenate is stable for a long time when stored at 4 ° C.
On the other hand, protease-incubated tissues lose potency almost immediately and return to control levels in a few days. Third, incubation at 38 ° C. for 2.5 h before repeated incubation at 38 ° C. for 2.5 h in the presence of protease appears to inhibit the appearance of neutrophil activator (Author's Note), while Incubation of pancreatic homogenate at 38 ° C. for a prolonged period (4 hours or more) does not appear to have any effect on potency.

Neutrophil activation in response to incubation with rat pancreatic homogenates results in very high levels of in vitro neutrophil activation. This activation can be inhibited using an exogenous serine protease inhibitor. Futan is the most effective inhibitor. The mechanism of this inhibition is unknown, but CD4 on neutrophil membrane surface
It is thought to be due in part to the inhibitory effect of protease inhibitors binding to No.3. Application of sub-stimulatory levels of pancreatic homogenate or the serine protease trypsin or chymotrypsin to non-irritating tissue results in release of neutrophil stimulating factor by this tissue. This stimulator has not been clearly identified, but appears to be essentially different from the factor released by the pancreas. The pancreas, through the release of endogenous neutrophil activators and proteases that promote neutrophil activators in other tissues,
It may be a major source of neutrophil activators in the body.

7.5 Pancreatic Activator and Neutrophil Response to Shear Stress In Vitro 7.5. A Introduction The purpose of this experiment was to provide further in vitro evidence of the stimulatory effect of filtered pancreatic homogenate on neutrophils by observing the factor inhibitory effect of neutrophil response to fluid shear stress. Leukocytes migrate from the hematopoietic pool across the medullary endothelium into the circulation and, under inflammatory conditions, from the circulation across the endothelium to the site of inflammation.
These movements require the attachment of leukocytes to the endothelium and pseudopodia formation. Pseudopods (also known as microvillous pseudopodia) occur as protrusions on the cell surface and may appear on endothelial cells as well as on leukocytes. These overhangs are strongly associated with the formation (polymerization) of the F-actin network. The pseudopodia are stiffer than the major cell body, thus making it more difficult for circulating activated neutrophils to pass through the capillaries.

Fluid shear stress (〜1 dyn / cm ² ) has previously been shown to result in rapid retraction of pseudopodia of untreated human neutrophils. The mechanism involved in this response is unclear, but is thought to involve Ca ++ and K + flux, as well as cyclic GMP.

To further define the activity of new leukocyte activators that occur in the pancreas, the effects on human neutrophils on the shear stress response were examined.

[0356] 7.5. b Method In these experiments, shear stress was kept sufficiently low (〜1 dyn / cm ² ) to prevent significant viscoelastic cell deformation. Fresh leukocytes from blood pierced by a healthy volunteer's pin were collected in a vacutainer tube (sodium heparin pretreatment, Becto
n Dickinson, Franklin Lakes, NJ). Red blood cells were sedimented at room temperature for about 30 minutes. Platelet and leukocytes are charged with a portion of the supernatant mixture by Plasma Lyte (Baxter Healthcare).
Corp., Deerfield, IL). Neutrophils, monocytes and lymphocytes were identified by morphology (125 × magnification). All experiments were performed within 4 hours with the following blood draws.

Rat and porcine pancreatic homogenates were collected as described in Example 7.2 (more specifically, Example 5). A low molecular weight aliquot was cut at 3 kD and filtered as described above. 100 μl of the cell preparation was transferred to an inverted microscope with a 50 × objective (Leitz Dievert).
, Germany) into a small chamber with a clear bottom. The microscope light source had a thermal filter and all experiments were performed at room temperature. The microscope eyepiece was replaced with a 25 × objective lens, analog background subtraction (Model
LKH 9000, LK Hawke Inc., Research Triangle Park, NC), Video Timer (
Model G-77, Odetics, Anaheim, CA), VHS video cassette recorder (Model
AG 1270, Panasonic, Japan) and a black and white combined charger camera (Model JE2362, Javelin, Japan) with monitor (Model VM 4512, Sanyo, Japan) connected to the closed circuit TV system.

The micropipette was assembled using a micropipette puller (David Koph Instruments) (range 1-3 μm id). The micropipette was connected to a reservoir with a water pressure regulator. The attached leukocytes spreading on the glass surface were identified and a single micropipette was placed on the cells so that a jet of fluid could be applied to the surface. The micropipette was tilted about 30 ° from the surface and the tip of the pipette was 5 μm from the cell surface. Numerical calculations (see Trapali et al. (1996) Life Sci 59: 849-857) show that the centerline velocity of the fluid jet outside the pipette tip is 0.74 m / s and the shear stress on the cell surface is 0.7. The range was from 02 dyn / cm ² to 0.4 dyn / cm ² .

[0359] 90 μl of the cell suspension was placed in a small chamber. Thereafter, 10 μl of pancreatic homogenate was added to the cell suspension. In the futan experiments, 10 μl of pancreatic homogenate and 10 μl of various concentrations of futan were added to 80 μl of cell suspension. For each observation, one isolated activated neutrophil was selected and photographed for 7 minutes. Shea
Stress was applied 3 minutes after 2 minutes.

[0360] 7.5. c Results The photographed neutrophils showed various responses to shear stress. For the purpose of measuring the amount of results obtained, the neutrophil responses were classified into three categories: • Complete response: The neutrophils contracted all pseudopodia during the application of shear stress and formed a circular shape. Pretend to be Protrude again after stopping the flow. -Partial response: This neutrophil can be retracted by part of the pseudopodia, but not all, and does not release new pseudopodia. Non-response: This neutrophil does not contract any pseudopodia. For different groups of cells incubated with pancreatic homogenate, 3
The proportion of different types of responses was determined. Rat 1 and rat 2 were treated with filtered pancreatic homogenates from two different rats. Pig 1 Low MW and Pig 2 Low MW were treated with a low molecular weight fraction (<3 kD) of pancreatic homogenate from two different pigs. Pig 2 (whole) was treated with whole pancreatic homogenate from the same pig as Pig 2 Low MW.

All control cells fully responded to sheer stress, whereas less than 20% of cells treated with pancreatic homogenate completely responded to sheer stress. 20-50% of the treated cells showed no response to shear stress. Although porcine pancreatic homogenate appeared to have a stronger effect than rat pancreatic homogenate, this observation needs to be established by further experiments. In a limited sample experiment (13 cells observed), no significant difference was found between the whole pancreatic homogenate and its low molecular weight fraction in suppressing the shear stress response. To study the mechanism by which pancreatic activators act on neutrophils, the moderating effect of the serine protease inhibitor inhibitor was measured. 10 μl of futan (0.05 and 0.13 mg / ml dissolved in 5% glucose) was added to 80 μl of cell suspension and 10 μl of porcine pancreas homogenate. Result is,
Futan at these concentrations was shown to have a slight pre-inflammatory effect on neutrophil activation, as in the assay according to this test; None showed a complete response.

[0362] 7.5. d Discussion Pancreatic homogenates from either rats or pigs partially suppressed the normal response of untreated human neutrophils to shear stress in vitro. This indicates the presence of one or more neutrophil activators present (possibly even at high molecular weight) in the low molecular weight fraction (<3 kD). Contrary to the results of in vitro pseudopodia formation, futan did not appear to downregulate neutrophil activation by pancreatic homogenates as measured by response to shear stress. The reason for this lack of inhibition is unclear, but may be caused by the low pH of the soluble phthalane (see Example 8).

Example 8 Beneficial Effects of Serine Protease Inhibitors on Visceral Arterial Occlusion (SAO) Shock Summary tetrazollum) (N
As a result of BT) and pseudopodia activation tests, it results in unregulated levels of leukocyte activation. Also, homogenates from pancreas, not from other tested tissues,
These same tests will activate untreated neutrophils. This activation is
Serine protease inhibitors, especially futan (nafamostat mesylate),
Was partially suppressed by the application of.

This activation was confirmed to be suppressed in vivo. Randomly selected rats were weighed, anesthetized, and an intravenous catheter was inserted and used for blood pressure measurement and anesthesia, respectively. In addition, a second venous catheter was inserted and connected to an infusion pump that infused phthalane or the same volume of saline at a rate of 3.3 mg / kg body weight (hourly). One hour after the pretreatment period, an abdominal incision was made and the superior mesenteric and celiac arteries were
Ligation was performed for 0 minutes, after which the ligation was removed. Animals were observed 60 min after reperfusion until the survival or mean arterial pressure fell below 30 mm Hg. At the end of the experiment, arterial blood was drawn for determination of plasma peroxide concentration using a peroxide electrode assay.

The results show a significant difference in blood pressure after reperfusion between SAO shocked and futanized SAO shocked animals (P <0.005, all time points greater than 90 minutes); The number of surviving animals treated with futan is significantly increased compared to untreated animal controls (P <0.001). The peroxide levels of the futan-treated SAO shock plasma are also significantly lower (P <0.05) compared to those of untreated SAO shock animals. However, both values were significantly higher than the initial plasma peroxide levels (P <0.001). These results indicate that SAO shock can be mitigated by pretreatment with the serine protease inhibitor futan, and some of this protection can be obtained from the ability of the protease inhibitor, as measured by plasma peroxide formation, Figure 4 shows limiting circulating levels of activator during the shock period.

[0366] Factors in the pancreatic homogenate activated untreated neutrophils in vitro.
Because of the large involvement of neutrophils in SAO shock, it was hypothesized that activators produced by the pancreas were sufficient by themselves to stimulate neutrophils in vivo and were responsible for the state of shock. The serine protease inhibitor futan, as reported in previous studies, mitigates SAO shock in vivo,
Upregulation of untreated neutrophils exposed to the incubated pancreatic homogenate was suppressed.

The bolus administration of the incubated pancreatic homogenate was tested for the ability to induce circulatory shock in rats. The ability of futanine pretreatment to mitigate shock induced in this manner was also tested. The sham SAO shock protocol was repeated as previously described (either 60 min futan pre-treatment or saline pre-treatment, and a 2 ml bolus dose of either pancreatic homogenate or low molecular weight pancreatic homogenate injected at arterial ligation. ). Injection of whole pancreatic homogenate was immediately lethal in saline-treated controls, but recovered after short-term hypotension in rats treated with futan (P <0.001 intergroup blood pressure after injection). Repeating the 3 ml experiment with the low molecular weight pancreatic homogenate resulted in a transient decrease in blood pressure for the homogenate (P <0.001 compared to the initial pressure), and the animal subsequently recovered.

For the purpose of studying the physiological activity of pancreatic homogenate on in vivo microcirculation,
Fluorescent in vivo preparations used rat mesentery and were perfused with pancreatic homogenate or control buffer. Perfusion of pancreatic homogenate resulted in a marked increase in the index of hydrogen peroxide formation in DCFH neutrophil fluorescence. Propidium iodide fluorescence (used in the hydrogen peroxide index to measure cell death) increased, but was not significantly different from the increase in control animals. Perfusion of whole pancreatic homogenate also resulted in a marked increase in neutrophil adhesion and microcirculatory vascular compression. These results suggest an in vivo role for bioactive factors released from the pancreas in shock and other pathological events.

8.1 Introduction Neutrophil activation is performed by applying homogenates tested in other organs,
The in vitro was performed applying pancreatic homogenate. This activation was suppressed by different serine protease inhibitors. Futan has been found to be the most effective. Visceral artery occlusion (SAO) shock is a shock model that targets the visceral area, particularly the pancreas, and causes systemic up-regulation of neutrophils.

Thus, in this model, in vivo systemic neutrophil activation due to inadequate release of neutrophil activators, partially derived from the pancreas, may appear. This study sought to determine whether the detrimental effects of SAO shock could be mitigated by futanine intervention in vivo.

The release of pancreatic components may be an important phenomenon in neutrophil activation and shock pathogenesis. Due to the presence of neutrophil activator in the pancreas, it produces neutrophil activator (Example 7), as well as high concentrations of serine protease, and the pancreas has sufficient concentrations of activator and other It has been hypothesized to contain toxins that cause acute shock without involvement of stimuli. The SAO shock experiment described in Example 7 was repeated using bolus administration of pancreatic homogenate, mimicking the release of splanchnic arteries and the release of pancreatic contents seen with SAO shock. Because of the ability of the serine protease futan to mitigate neutrophil activation in vitro, it was hypothesized that futan pretreatment was effective in reducing the effects of bolus administration of pancreatic homogenate.

In addition, for the determination of low molecular weight neutrophil activator in pancreatic homogenate, we are interested in determining whether this factor or other co-existing constant molecular weight species is sufficient to induce shock in rats. was there. The mimicking SAO shock was repeated with a low molecular weight pancreatic homogenate bolus dose mimicking the release of arterial ligation.

Following the study of pancreatic homogenates in neutrophil function in vitro and the in vivo response of all animals, it was of interest to determine the physiological mechanisms of pancreatic homogenates that cause shock in vivo. To understand this bioposition, in vivo fluorescence microscopy preparations were tested on rat mesentery to observe the effect of filtered exogenous pancreatic homogenate.

8.2 Method 8.2. a Method: SAO shock FUTHAN pretreatment experiment The method is a modification of Example 4 described. Male Wistar rats (250-320 g) were randomly divided into SAO shock (n = 7) and SAO shock sham groups (n = 6). Animals were under general anesthesia using pentobarbital (50 mg / kg im) and cannulated via the femoral artery and vein. Heparin was not injected except as required to ensure an open catheter line (10 U / ml Plasma-Lyte). One femoral artery is connected and continuous mean arterial pressure (MAP
) Was measured. On the other hand, the rest was used for collecting blood samples. Insert a second venous catheter, connect to the infusion pump, and add the same volume of 3.3 mg / vol.
The injection was performed at a rate of kg body weight (every hour). MAP and heart rate were recorded. Preliminary experiments used a small bolus dose of futan at concentrations ranging from 1-20 mg / kg body weight (hourly) instead of an infusion pump. One hour after the pretreatment time, an abdominal wall incision was made and the superior mesenteric artery and celiac artery were ligated for 90 minutes, after which the ligation was removed. Animals were observed for survival after 60 minutes of reperfusion or time until mean arterial pressure fell below 30 mmHg.

At the end of the experiment, the arterial blood was drawn and the plasma peroxide concentration was measured using the peroxide electrode measurement technique of Example 2 above.

[0376] 8.2. b Method: Pancreatic homogenate infusion experiment The SAO shock experiment with bolus administration of pancreatic homogenate was repeated to replace the "unclamping" of arteries and the pancreatic components into circulation instead of actual ischemia / reperfusion of visceral areas. Mimicking release. The low molecular weight (2 ml) as well as the whole homogenate (2 ml) were tested for their ability to induce hypotension and circulatory shock. The ability of futan to counteract the infusion effect of pancreatic homogenate also
Tested with shock protocol. At the end of these experiments, the arterial blood was drawn and the plasma peroxide concentration was measured using a peroxide electrode measurement technique.

[0377] Futane, or the compound nafamostat mesylate (6-amidino-2-naphthyl)
-p-guanidino-benzoate (dimethanesulfonic acid) is a low-molecular-weight serine protease inhibitor manufactured by Torii Pharmaceutical Co., Ltd. (Tokyo, Japan). , Saitama, Japan. (See Example 7).

[0378] 8.2. a Method: In Vivo Fluorescence Microscopy Experiment An in vivo fluorescence microscope for the preparation of rat mesentery is described in Example 3 previously. The perfusate reservoir is evacuated and connected directly to a perfusion pump that can be adjusted to provide a variable flow rate to the mesentery. After collecting from the dispensing stage to the reservoir, recirculate. Alternatively, the liquid is circulated in the bypass circuit without the perfusion stage.

This protocol is based on Plasma-Lyte, a physiological buffer that does not require continuous nitrogen degassing (Upjohn Comp., Kalamazoo, M.L.) instead of Krebs-Henseleit perfusion buffer.
Modified to use l). To enable mesenteric perfusion with a limited volume of purified pancreatic homogenate, a recirculating drip system was devised to allow continuous perfusion of pancreatic homogenate. The Plasma-Lyte was kept in a reservoir (60 ml) under negative pressure, connected to a syringe with a polyurethane tube, and the perfusate was pumped through a 3-way cock to either fluid recirculation or conditioned perfusion. After perfusion of the mesenteric preparation, the perfusate was drained through specially drilled holes to the animal stage and the fluid was returned to the reservoir. The animal stage was segmented to prevent mixing of the perfusate with the body fluid of the animal. The animal was lifted from the floor to the stage and the animal effluent was transported under negative pressure to a waste container.
Rats with peritoneal blood or bleeding from an abdominal wall incision were not used.

After a 10 minute stabilization period, mesenteric preparations were treated with propidium iodide (PI) (1 μm).
m) (Sigma Chemical Co., St. Louis, MO) and dichlorofluorescein diacetate (DCFH-DA) (10 μm) (Molecular Probes, Eugene, OR) was added to the reservoir and selected tissue sections Background autofluorescence was recorded.
Initial recordings were taken with bright fields (40 × water-immersion, Leitz) and fluorescent images (20 μm-100 μm) of selected venules and arterioles. Five to six observation fields were randomly selected and brightfield, PI, and DCFH recordings were recorded every 20 minutes with a CCD camera coupled to a video cassette recorder. Images were recorded for later analysis. Fluorescent light exposure time was minimized to avoid whitening of the pictures.

10 minutes after the first recording, 3 ml of filtered pancreatic homogenate was added to the reservoir. 500G centrifugation, followed by filtration through filter paper (0.78 μm vacuum filter (M
A filtered homogenate of illipore Filter Co., Beverly, MA) was required to prevent opacity that would be due to the presence of large solutes. After 10 minutes, a secondary recording was prepared and recording was continued every 20 minutes for 120 minutes, after which the experiment was terminated.

Videotapes were played for analysis of cell death as measured by PI and hydrogen peroxide production, measured by DCFH. Analytical venules were limited to 20-80 μm in diameter. P
The number of I-positive cells was calculated in the initial time section of 5-6 randomly defined areas in the mesentery and collected every 20 minutes. The entire field of view was used at approximately 300 μm × 300 μm for this purpose. The number of dead cells was compared at different times, DCFH fluorescence was recorded along the entire length of the relevant venule throughout the experiment and compared to background fluorescence in stroma (NIH image and Adobe Photoshop software package) . DCFH fluorescence was compared for 20 minutes during the experiment. In addition, leukocyte fixation and vessel diameter were recorded throughout the experiment. Leukocytes were counted as the average number of stationary cells for 30 minutes. At a defined location on each recording vessel, the vessel diameter was measured, arbitrarily selected, and displayed as a normalized average taking into account different vessel diameters. Lengths were calculated using the NIH Imaging Software Package, comparing to standards.

Statistical analysis of the significance of differences in DCFH fluorescence, PI cell death, vessel diameter and leukocyte adhesion was measured as Student's t distribution and presented as the mean of standard deviation.

8.3 Results The SAO shock was associated with the 90 ligation of the superior mesenteric and celiac arteries described in Example 4.
Removal at one minute resulted in consistent hypotension and death. Initial attempts to prevent death following hypoperfusion and subsequent reperfusion with low dose bolus (0.05 ml) futan failed. Concentration 1 (n = 3), 3.3 (n = 3) with pretreatment either 60 minutes before shock (1, 10 and 20 mg / kg) or 30 minutes before occlusion (3.3 mg / kg) Infusions of 10 (n = 3) and 20 (n = 2) mg futan / kg body weight every 5 minutes failed to uniformly restore MAP or prolong survival. Similarly,
In these initial experiments, futan was ineffective at reducing the increase in plasma peroxide formation seen after SAO shock. There were no significant differences between the control and the futan-treated groups (P <0.005 both groups, after SAO shock (n = 9) and before SAO shock (n = 7)).

[0385] The reason for the ineffectiveness of phthalane in improving SAO shock in early experiments appeared to be due to the pH of futan in solution. This pH is about 3.5 at a concentration of 1 mg / ml in 5% glucose-deionized water (D1) according to the manufacturer's instructions. In the established protocol, about 2 ml of this solution was added at a rate of 0.05 ml every 5 minutes,
The blood was injected into a 20 ml (approximate) rat blood volume. In sham-shocked animals (not reported herein), a buffering volume of blood is able to absorb the pH disturbance.

However, in shocked animals, the pH buffering capacity (particularly the pancreas and small intestine) is severely impaired. Therefore, injection of futan may contribute to irreversible acidosis. Injection of 0.05 ml volume of futan resulted in a transient hypotensive state. Animals recover from these MAP transients, but animals injected with futan generally showed lower MAP than saline injected groups. This difference was not significant. Futan includes alcohol, rat serum, DMSO, and physiological pH.
Was found to be insoluble in Trisma buffer.

The problem of low pH and antihypertensive effects of futan was that at the initial rate (futan was infused at a concentration of 3.3 mg / kg body weight / hour via the infusion pump), high Concentration of phthalane (20 mg / ml) at very low flow rates, effective injection concentration of 3.3 mg
/ kg bw / hr, alleviated by continuous infusion of futan. In this way, the injection volume was reduced to less than 1 ml (about 0.8 ml), minimizing the potential for acidosis. Care must be taken to ensure that this concentration (20 mg / ml) does not crystallize in the catheter.

The 60-minute futan pretreatment using this modified protocol resulted in a beneficial decrease in blood pressure drop at all times after arterial ligation removal (P <0.005). Futan prevented the death of futan pretreated animals after SAO shock at the end of the 30 minute reperfusion time and also improved survival time. No saline-treated control animals were alive until this time.

In addition to improving blood pressure and decreasing mortality, futan-treated rats have significantly more circulating peroxide production compared to control animals after SAO shock, as measured by a plasma peroxide assay. Low level (P <0.05). There were no significant differences between the groups before shock treatment. Despite the reduction in the futan-treated group compared to the post-SAO-shock control, it was not possible for futan pretreatment to prevent an increase in peroxide production after the SAO shock. Compared to circulating values before the shock protocol, the futanized and saline-treated control groups after SAO shock were:
Circulating peroxide production was significantly higher (P <0.005).

Deaths in the saline-treated group were almost immediate, with the exception of one animal that survived for some time. In the group treated with futan, none of the animals died during the observation period after the injection for 60 minutes, and the MAP recovered to almost the initial value. Futan pretreatment with an infusion pump at a concentration of 3.3 mg / kg body weight results in a decrease in MAP as described above.
Injection of low molecular weight pancreatic homogenate was not lethal in any animal experiments (n = 4).

8.4 Discussion Intravenous injection of futan mitigates the production of circulating peroxide in SAO shock. Futan pretreatment increases systemic blood pressure and survival time for SAO shock.

Example 9 Isolation studies on pancreatic homogenates Summary The discovery of neutrophil activators from the pancreas prompted a search for the identification of these substances. From the pancreas's unique position as a source of catabolic digestive proteases and zymogen precursors, proenzyme peptide remnants or other small degradation products from the pancreas
It appears to be able to function as a low molecular weight (<3 kD) neutrophil activator. The pancreas is a source of other low molecular weight species that have been shown to be produced in the pancreas and may also be involved in the up-regulation of neutrophils in vivo, especially platelet activating factor (PAF) and PAF-like substances is there. Low molecular weight rat pancreatic homogenates were separated by FPLC and high performance liquid chromatography (HPLC) fractionation and analyzed by mass spectrometry.

Although no definitive identification was performed, the activator appears to have a molecular weight of 300-800D. Activators are probably composed of several different factors.

9.1 Introduction In vivo and in vitro experiments have shown that the effects of neutrophil activators can be inhibited by the application of serine protease inhibitors, most particularly phthalane. These results indicate the involvement of pancreatic digestive proteases as neutrophil activators. Inhibition of pancreatic homogenates by futan in vitro may be due in part to inhibition of neutrophil activation of neutrophils themselves. In vivo protection by futanes against neutrophil activation can be achieved by stabilization of pancreatic lysosomes and acinar cells, as well as by direct down-regulation of neutrophils. Recovery of neutrophil activating activity in shock plasma and the low molecular weight fraction of pancreatic homogenate indicates that other factors are involved. A systematic method was performed to identify and / or eliminate potential (particularly low molecular weight) factors from the pancreas that could function as neutrophil activators.

9.1. a Peptides From the enormous amount of proteases in the pancreas, often in zymogen form, it seemed reasonable to infer these factors as neutrophil activators. In particular, neutrophil activation by the low molecular weight (<3 kD) pancreatic homogenate fraction indicates that the activator may be one of many propeptides that are cleaved upon activation of the major protein. Was. In addition to the many different proenzymes in the pancreas that can result in multiple peptide products, there are also many different proteolytic enzymes that can be involved in proenzyme peptide cleavage. Each of these proteolytic enzymes preferentially cleaves various sites in the amino acid chain, resulting in a vast number of possible peptide sequences. This causes excessive peptide substitutions that would be very difficult and costly to analyze individually. To assist in the identification analysis of potential peptide activators, a computer program was created to analyze various potential peptide substitution products and compare them to estimated molecular weights determined by mass spectrometry.

9.1. b Platelet activating factor (PAF) and PAF-like substances Platelet activating factor (PAF) is a small amphiphile that is known to mediate a wide variety of biological effects at concentrations as low as 10 ^-10 M. (1)
). PAF in vitro aggregates platelets, is also chemotactic for neutrophils, and is a mild inducer of respiratory burst (see Example 6). PA
Infusion of F caused hypotension and shock in experimental animals and caused acute pancreatitis when injected into the rabbit upper pancreatoduodenal artery. PAF has been implicated in the pathology of various disease states, such as sepsis and shock. In particular,
PAF has been postulated to be a major factor in the course of visceral artery occlusion (SAO) shock. Although PAF is thought to be involved in neutrophil activation, as measured in pancreatitis, one study failed to find evidence of PAF in the acute state. The pancreas has also been shown to produce PAF in vitro, as do many other tissues in response to stimuli.

The results obtained from the application of PAF antagonists in inflammatory conditions are mixed, and the clinical role of PAF in inflammation is still uncertain. Some inventors have found plasma levels of PAF or lyso-PAF (an indirect indicator of PAF concentration) and have clinically reduced patients who actually suffer from sepsis and other diseases. Whether the PAF actually circulates or remains bound during various pathological (ischemia) events, and also whether the PAF functions as a key mediator or interacts with other activators. It is also unclear whether it is a subordinate to respond.

Platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) has an O-alkyl ether group at sn-1 and an acetate group at sn-2. And a glycerol backbone with phosphocholine at the sn-3 position. About 95% of the PAF compounds have 16 or 18 carbon saturated chains in the ether bond of sn-1. Unsaturated ether groups were detected, but show lower potency. Acetate groups are also important for PAF bioactivity, with increasing chain lengths of more than 3 carbons decreasing bioactivity. Hydrolysis to the hydroxyl group (HO-) at the sn-2 position causes the formation of lysoPAF, followed by a loss of biological activity. Lyso-PAF is the most important degradation product of PAF, and also its precursor, under inflammatory conditions (Mclntyre et al. (1995) Physiology and
Chapter 13 of the Pathophysiology of Leukocyte Activation, edited by Graner et al., Oxfo
rd Press, Oxford, pp. 1-30; and Anderson et al. (1991) Surg Gyne.
col Obstet 172: 415-424). PAFs can be formed fresh or by the reconstitution pathway (eg, Prescott et al. (1990) Thromb Haemo.
st 64: 99-103). The new synthetic route is P
This is a mechanism related to AF formation.

[0399] In response to inflammation, the remodeling pathway is stimulated. It is thought to be the major pathway of PAF production by inflammatory mediators (Anderson et al. (1991) Surg.
Gynecol Obstet 172: 415-424). Rebuilding path, first, by hydrolyzing fatty acyl groups of sn-2 Phospholipase A ₂ is alkyl choline phosphoglycerides glyceride (Prescott et al. (1990) Thromb Haemost 64: 99-10 3), forming a lyso PAF After that, it can be converted to PAF by the action of acetyltransferase. The PAF acetylhydrolase, in the opposite way to the phospholipase A ₂ which only cut only group of the short chain, degrades PAF (S
(1985) Adv Prostaglandin Thromboxane Leukot Res 15: 693-696; and Stafforini et al. (1997) J Biol Chem 272: 1.
7895-17898). In vitro, the PAF by phospholipase A ₂ sn
-2 and at sn-3 by phospholipase C (Mclntyre et al. (1995) Physiology and Pathophysiology of Leukocy).
te Activation, Oxford Press, Oxford, 1-30). A suitable concentration of PAF acetylhydrolase in vivo is probably less than 30 minutes for PAF in plasma.
Short half-life. PAF acetylhydrolase from plasma can be oxidatively inactivated, a scenario that may be of physiological significance in reperfusion injury. Organ and pancreatic homogenates activated by serine protease, tissue PAF acetylhydrolase activity,
It is also sensitive to trypsin cleavage. However, PAF acetylhydrolase in plasma is resistant to trypsin treatment. The mechanism for the production of PAF-like substances in homogenates activated by serine proteases, including pancreatic homogenates, can lead to an increase in PAF and PAF-like substance concentrations, with the degradation of PAF acetylhydrolase. It is not known whether plasma PAF acetylhydrolase is sufficient to block the possible formation of PAF-like substances from inappropriate concentrations of circulating proteases.

[0400] PAF-like substances are small lipids whose vasoactivity is very similar to that of PAF. Although these substances often tend to be orders of magnitude less active than PAF, they function in the same way by binding to the PAF receptor and co-localize in thin layer chromatography (TLC). Become Given these similarities, reports intending to measure PAF inhibition by inhibitors or PAF concentrations by bioassay can be measured without the awareness of PAF-like substances instead. This is an important feature, presumably because PAF-like substances are obtained from an oxidative mechanism rather than through an enzymatic pathway. A significant difference, especially in diseased states, is that production of PAF is tightly regulated, whereas PAF-like substances are unregulated inflammatory products. Standard product P
AF remains bound to the endothelium even when produced by inflammatory mediators such as high concentrations of hydrogen peroxide (1 mM). In contrast, PAF-like substances have a lower endothelium due to lower concentrations of H ₂ O _{2 for} longer (at least one hour), or tert.
Occurs when exposed to fat-soluble peroxides such as -butyl hydroperoxide (t-BuOOH). Endothelial cells treated with t-BuOOH produce numerous membrane vesicles in response to oxidative stress. These vesicles appear to resemble those seen in vitro when neutrophils are incubated with pancreatic homogenates.
The production of endothelial bleb formation can be blocked in vitro by the application of free radical scavengers, providing further evidence of an oxidative mechanism for their formation (Mclntyre et al. (1995) Physiology and Pathophysiology of Leuko).
cyte Activation, Oxford Press, Oxford, 1-30). Like PAF, P
The position of the sn-2 and sn-3 of the AF-like lipid, respectively, is decomposed by phospholipase A ₂ and E Suhoripaze C. In addition, these materials also can be decomposed at the position of sn-1 by phospholipase A _1, rather than ether bonds of standard PAF indicates the presence of an ester bond at this position. PAF-like substances are thought to form by oxygen free radical-mediated cleavage of cell membrane components (phosphatidylcholine) at a number of positions at the sn-2 position of the unsaturated (arachidonate). (For a thorough discussion of the mechanism of lipid peroxidation mediated by oxygen free radicals, see Example 3, section 3.1.c Lipid peroxidation).

9.1. c Endotoxins Endotoxin leakage appears to be responsible for hemorrhagic and intestinal shock heart failure in dogs. There is much speculation about the effects of endogenous enterotoxin and the gram-negative bacterial peptide fMLP on the course of circulatory shock. Although it is generally agreed that endotoxin translocation plays a role in the pathogenesis of these conditions, the extent of its contribution is unclear.

In tests herein, larger endotoxins could be present in pancreatic homogenates of full molecular weight and in homogenates treated with proteases. The application of antimicrobial substances (see Example 5) and the discovery of low molecular weight activators, much smaller than endotoxin, was attributed to the fact that endogenous endotoxin was not treated with pancreatic homogenate and protease-treated homogenate found in these studies. Strongly indicates that it is not a category of neutrophil activator. The bacterial peptide fMLP was not found in any mass spectrometry.

9.2 Method 9.2. a Method: FPLC Separation To identify neutrophil activators present in the pancreas, rat pancreas was collected, homogenized and incubated as described in Example 5. The pancreatic homogenate was filtered by centrifugation at 500 G, and the filtrate was collected.
Ultrafiltered through a kD cutoff filter. Ion exchange high pressure liquid chromatography FPLC ™ (Gradient programmer GP-250, liquid chromatography controller LCC-500, Pharmacia LKB Biotechn)
(U.S.A., Upsala, Sweden) was used to isolate 100 μl of pancreatic ultrafiltrate. Samples were injected at a flow rate of 1 ml / min into either the MonoQ ™ HR5 / 5 or MonoS ™ HR5 / 5 columns. Buffer A of the MonoQ column is 20 mM
Tris-HCl (pH 8.0) and Buffer B was Buffer A + 1 M NaCl. Fractions were eluted using standard solute elution of 15 ml 0-35% buffer B, 10 ml 25-100% buffer B, and 5 ml 100% buffer B. MonoS elution was performed using the same profile, substituting 50 mM acetate-NaOH (pH 5.0) for buffer A instead of Tris-HCl. Aliquots of representative peaks were taken and assayed for neutrophil activation using the pseudopodia formation test described in Example 2. Although activation was demonstrated in the cation column as well as in the sample obtained from the anion column, the anion preparation showed a more defined elution profile and was used for subsequent purification by HPLC.

9.2. b Method: RP-HPLC separation FPLC MonoQ column showing substantial neutrophil activating activity (fraction # 2-3)
The purified pancreatic homogenate fraction separated by was subjected to reversed-phase high-performance liquid chromatography (RP-HPLC). 0.1% trifluoroacetic acid as buffer A
250 μl of sample was injected using (TFA) and 0.1% TFA + 80% acetonitrile as buffer B (Waters M-45, automated gradient controller, Lambda-Max 480LC spectrophotometer, Millipore Co.) ., Milford,
Ma). 4.5 ml of 0-10% buffer B, 10-40% buffer B 1.
5 ml, 40-45% buffer B 2.25 ml, 45-100% buffer B 5.25 ml
The fractions were eluted using a solute elution of 1.5 ml of 100% buffer B and a flow rate of 0.5 ml / min. The peak was detected by the increase in absorbance at 215 nm and the eluted fractions were collected manually. Before applying the fraction to untreated neutrophils for the pseudopodia formation test,
The purified homogenate containing the HPLC solvent was volatilized under nitrogen gas.

9.2. c Method: Matrix-Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry FPLC and HPLC fractions containing neutrophil activating activity were processed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. MALDI is a standard, well-known method of analyzing proteins and peptides that does not require extensive purification. The matrix used was sinapinic acid, a preferred matrix for samples containing a mixture of water-acetonitrile, such as those containing HPLC fractions.
cid) (trans-3,5-dimethoxy-4-hydroxycinnamic acid, MW 224D)
(See, for example, Beavis (1996) Methods in Enzymol 270: 519-551). Ultrafiltered (<3 kD) rat plasma collected before and after SAO was also measured by MALDI. Differences in rat shock plasma spectra were plotted using the MATLAB software package (The Math Works, Inc., Natick, Mass.).

9.2. d Method: Neutrophil PAF Inhibition Experiment Pancreatic neutrophil activators incubated with protease and their
To determine whether the tissue homogenate is associated with PAF,
Pase C (phosphatidylcholine choline phosphohydrase type XI: 3.2 M (NH _Four )_TwoSO_Four pH: Sigma Chemicals obtained from B. cereus suspended in 6.0.
St. Louis, MO), enzymes with non-specific PAF inhibitor properties, and
In addition, actin polymerization and superoxide can be performed using commercially available PAF inhibitors.
A sid formation test was performed. The PAF inhibitors used were 10 μM (±) -trans-2,5-bis (3,4,5-trimethoxyphenyl) -1,3-dioxolane (dioxolane) (Cal BioChem, San Diego, Calif.), And 5 μM, 50 μM
And WEB2170 (Boehringer Ingelheim, Germ) at a concentration of 500 μM.
any). The concentrations of phospholipase C used were 0.2 U / ml, 1 U / ml, and 2 U / ml and did not interfere with neutrophil activation (Wazny et al. (1990) Eu
r J Clin Microbiol Dis 9: 830-832; Lin et al. (1997) Respir.
64: 96-101; and Styrt et al. (1989) J Lab Clin Med 114: 51-55). Phospholipase A also degrades PAF_Two(Sigma Chemicals, St. Louis, MO, obtained from calf pancreas) was assayed (concentration: 1 U /
ml, 10 U / ml, 20 U / ml) were unacceptable due to their neutrophil irritancy (
Hazlett et al. (1990) Adv Exp Med Biol 279: 49-64; Langholz.
(1990) Prostaglandins Leukot Essent Fatty Acids 39: 227.
-229; Cicala et al. (1993) Gen Pharmacol 24: 1197-1202).
.

A rat pancreas homogenate was prepared as described in Example 5 and used for liver, spleen,
Intestinal and other organ homogenates of the heart were prepared by incubation with trypsin (1300 U / ml homogenate) or chymotrypsin (52 U / ml homogenate) as described in Example 7. PAF inhibitors were incubated with the tissue homogenate for 30 minutes at 37 ° C. Phospholipase C was incubated with the tissue homogenate for 10 minutes at room temperature.

Using 1 ml of filtered homogenate in the presence or absence of phospholipase C, lucigenin-enhanced superoxide production from human donor plasma (neutrophil concentration 150 × 10 ³ cells / ml) was used. Measured as described in detail in Example 6.

9.2. e Method: Peptide Sorter Computer Program In order to identify fractions isolated from the pancreas, a computer program was created to analyze various potential peptide substitution products and to determine their estimated molecular weights as determined by mass spectrometry. Compared. The program created by FORTAN77 reads amino acid sequences and compares them for homology with unknown substances that can be read and compared. The program was modular and menu-driven for easy modification and access. The user can enter the deduced peptide sequence, known mass, and search for similar peptides from certain species or search for the same peptide, and the program will enter the possible peptide mass, Flip out peptides that are close to mass or the desired species. When entering the deduced peptide sequence, after analysis, the program gives the user the option to add the peptide to the database. In this method, each successive addition to the list of potential peptide contributions is compared to those already listed to avoid duplication. When searching by molecular weight, a centroid for the molecular weight is calculated, and if this is changed by the user, other peptides having a molecular weight within the specified range can be found. Finding peptides that match the putative neutrophil activator molecular weight is a long and literature-dependent method. Any transposition proposition, ie, a peptide sequence that matches the putative molecular weight of the activator, will result in the identification of hundreds of peptide sequences, which cannot be tested individually. A table of the peptides tested is given in FIG. Most of the peptides tested were of pancreatic origin, but also include other ubiquitous peptides (eg, bradykinin, fMLP). These peptides were analyzed stepwise for similarity to putative neutrophil activators, not only for the complete peptide sequence, but also for its amino acid composition, along with the length of the peptide.

9.3 Results 9.3. a FPLC Elution Results Full molecular weight and low molecular weight rat pancreatic homogenate FPLC through a cationic MonoS ™ column resulted in the majority of the sample eluting into the first four fractions. Most of the neutrophil stimulating activity was also found in these four fractions, as assayed by actin polymerization and superoxide formation tests. In particular, fraction # 3 showed significantly better activation in both tests than the other fractions in the whole pancreatic homogenate sample, whereas fraction # 1-3 in the low molecular weight sample resulted in The highest degree of activation occurred in descending order.

Elution of the pancreatic homogenate through the FPLC anion MonoQ ™ column showed much better resolution than seen on the cation column, and the pancreatic factor was essentially uncharged or slightly cationic. It was shown. F
The pancreatic homogenate injected on a PLC anion MonoQ ™ column also showed, in total and low molecular weight fractions, a wider degree of neutrophil activation properties than that seen with the cationic column fraction.

In the whole pancreatic homogenate fraction (n = 3 pancreas), the highest degree of actin polymerization resulted from neutrophils treated with elution from fraction # 1-6, which was generally the highest. Superoxide production levels are also consistent with the indicated fractions. Fractions # 8-10 are not as stimulatory for neutrophils as can be demonstrated by the actin polymerization test, but are atypical initial increases in superoxide production, followed by much more than subsequent control values And low attenuation. The cause of this response is not known, but indicates the presence of additional activators with activation responses that are different from those typically measured (see Example 6). The low molecular weight anion column fraction (n = 3 pancreas) showed the highest degree of neutrophil actin polymerization in the first six fractions, consistent with the results obtained from the full molecular weight homogenate. Superoxide production is greatest in fraction # 2-7, which is consistent with the activation seen by the pseudopodia formation test for low molecular weight pancreatic homogenates. The early stage superoxide production seen in fractions # 8-10 of the whole homogenate elution was not detected in the low molecular weight fractions, and the source of superoxide production in those fractions was the higher molecular weight product. It was shown. The small increase compared to the control neutrophil pseudopodia formation seen in the higher numbered fractions (10-14) obtained from the cation and anion column FPLC elution is almost certainly an increase in the solvent salt concentration. , Which can cause indefinite activation. This guess is
Supported by superoxide measurements that were uniformly low in these eluted fractions in all pancreatic samples tested.

[0413] 9.3. b Reverse phase-HPLC elution results Fraction # 2 obtained from one of the low molecular weight anion FPLC column separations was further separated by RP-HPLC fractionation and these samples were tested for actin polymerization pseudopodia formation. Was measured for their ability to activate untreated neutrophils. An enormous amount of activity occurred in later fractions (# 16-18). The solvent eluted at this point is a strong detergent irritant for neutrophils.
00% buffer B (0.1% TFA + 80% acetonitrile), but removed most of the harmful components of the medium before volatilizing with nitrogen gas. This occurred as evidenced by the lack of neutrophil activation observed in sample # 15,
This shows a large HPLC elution peak at 100% buffer B, but does not activate the untreated cells appreciably.

[0414] 9.3. c Mass Spectrometry Results MALDI mass spectrometry was obtained from low molecular weight rat shock plasma and was isolated from rat pancreas homogenate FPLC and rat homogenate separated HP
LC was indeterminate for most of it. Mass spectrometry of low molecular weight rat shock plasma was obtained before and after the shock showed similar spectra. However, a direct comparison of the two spectra obtained from the two plots of the isolated segment showed some differences. In the absence of a focus at a particular molecular weight, it is difficult to interpret the myriad differences between the spectra of plasma collected before and after shock.

The mass spectrum of the low molecular weight pancreatic homogenate from FPLC MonoQ elution peaks # 2 and # 3 was analyzed. These two peaks show strong homology with each other, especially with a continuous molecular weight peak between 611-696D, with two additional simultaneous peaks at 471 and 879. Given the large number of possible peaks, it was difficult to interpret whether these peaks were related to the mass spectrometric mass of the shock plasma measurement. Then, MALDI mass spectrometry was performed on RP-HPLC fractions (peaks # 16 and # 17) obtained from the filtered rat pancreatic homogenate FPLC fraction # 2-3, which showed neutrophil activating activity. Went. These spectra show similar molecular weight peaks (to each other), which partially match the peaks found in the rat plasma spectrum, but do not necessarily match the peaks obtained from FPLC mass spectrometry measurements. . Peak # 17 eluting with 100% buffer B represents an ordered set of molecular weight peaks between about 991-1607D, which is not found in peak # 16, which elutes immediately before. This appears to be a series of detergent peaks associated with the HPLC and is not interpreted as a signal.

Includes high neutrophil activating activity levels, but elutes faster on HPLC columns,
The MALDI mass spectrum of the second HPLC assay fraction (fraction # 7 of 14 fractions) shows an accurate spectrum. It is unclear why the molecular weight peak of this pattern is not found in the mass spectrum of HPLC fraction # 16, which may be as a detergent, and the chemical is ubiquitous. I do. Since these peaks are not present in the FPLC mass spectrum, they may be artifacts of HPLC.

[0417] 9.3. d PAF Inhibition Results Initially, incubation of non-irritating organ homogenates of the heart, liver, spleen, and intestine with the serine protease trypsin or chymotrypsin
As described in Example 7, it caused a significant increase in pseudopodia formation as compared to controls. As indicated above, the pancreatic homogenate also substantially activated neutrophils. Application of phospholipase C, a non-specific PAF inactivator, caused a reduction in% pseudopodia formation by trypsin-activated homogenate, with a marked decrease seen in activated spleen and liver. Pancreatic homogenate-induced neutrophil activation was reduced by more than a third with minimal effect on trypsin-activated cardiac homogenate. Attempts to inhibit the effect of activated homogenates on neutrophil pseudopodia formation by the application of the commercial PAF inhibitor dioxolane have proven to be less effective.

To determine whether phospholipase C inhibition of neutrophil actin polymerization by activated homogenates also reduces superoxide production in response to these stimuli, plasma enhanced chemiluminescence with lucigenin was determined. Tested. These tests confirm the results obtained by the pseudopodia formation assay when the steady state chemiluminescence of the homogenate incubated with trypsin was reduced by the addition of phospholipase C. Plasma chemiluminescence of spleen and heart homogenates incubated with trypsin was reduced as compared to control values, in contrast to the neutrophil pseudopodia formation assay. The reasons for this decrease are unclear.

A third PAF inhibition experiment was performed to determine the change in neutrophil pseudopodia formation in response to trypsin-activated (n = 2) and pancreatic (n = 2) homogenates.
The measurement was performed in the presence or absence of WEB2170, which is an F inhibitor. 5 μM (n = 2), 50
The results obtained from application of WEB2170 at concentrations of μM (n = 2), and 500 μM (n = 2) indicate that neutrophil activation was reduced in response to pancreatic homogenates or homogenates incubated with trypsin. Proved almost ineffective.

9.4 Discussion Investigations for neutrophil activators present in plasma after inflammatory conditions such as shock, and for neutrophil activators obtained from the pancreas have isolated the factors. And ultimately led to an attempt to identify it conclusively. The plasma concentration of such activators is very low, and may be contaminated with other factors, including known activators at a later stage, such as cytokines. Thus, the concentration was aimed at isolating neutrophil pancreatic factor, possibly present at much higher concentrations and limited only by the amount of tissue homogenate available. Isolation of these factors in the pancreas has not been straightforward. From the results of mass filtration, it is likely that the neutrophil activator is present in the pancreatic homogenate at a (higher) molecular weight, and also at a low molecular weight of 3 kD or less. Focusing on low molecular weight activators,
There appear to be a number of factors that can activate neutrophils from the pancreas in vitro. Due to the unique proteolytic properties of the pancreas and the nature of species of generally lower molecular weight,
We focused on two groups of potential activators that could be present in the pancreatic homogenate.

The first of these are peptide fragments, which are a possible group of activators due to their low molecular weight and efficacy. Short peptides have been shown to activate neutrophils; the most famous group is the fMLP group obtained from bacteria (Schiffmann et al. (1975) Proc Natl Acad Sci USA 7
2: 1059-1062). Recently, three peptide fragments released from plasma in response to alkali have been isolated, which activate neutrophils in vitro.
(Pfister et al. (1993) Invest Ophthalmol Vis Sci 34: 2297-2 304; and Pfister et al. (1996) Invest Ophthalmol Vis Sci 37:
230-237). Pancreatic peptides are known to circulate in shock and other medical conditions (Merriam et al. (1996) J Surg Res 60: 417-421; Katz et al. (1964) Archives of Surgery 89: 322-331; Fo).
(itzik et al. (1995) Dig Dis Sci 40: 2184-2188; Leffler et al. (
1973) Am J Physiol 224: 824-31; Glenn et al. (1971) Cir.
c Res 29: 338-49; Lefer et al. (1970) Circ Res 26: 59-6.
9; Herva et al. (1970) Scand J Gastroenterol Suppl 8: 44-52;
(1970) Am J Physiol 218: 1423-1427) and thus may be responsible for systemic neutrophil activation in shock.

Thus, in an attempt to characterize the components of the partially purified pancreatic homogenate, a review of the literature has primarily focused on truncated and released in the pancreas in trauma or in response to other stress conditions. The procedure was performed on the resulting pancreatic peptide, especially the proenzyme fragment. A computer program was created and the number and sequence of these amino acids were analyzed to determine if they corresponded to a known neutrophil activator as determined by molecular weight analysis. To take full advantage of this capability,
First, the absolute molecular mass of the unknown activator must be measured.
MALDI mass spectra were performed on rat shock plasma before and after shock to determine the molecular weight of unknown neutrophil activators. While the number of large differences between the spectra before and after shock was negligible, the number of smaller differences in the mass spectra was so large that this proved to be the true method. Therefore, it is difficult to make a definitive measurement by this method unless the activator is known a priori or is not present at sufficient concentration.

To follow another method, rat pancreatic homogenates were obtained, filtered through a 3 kD cut-off filter, and separated by FPLC followed by HPLC. These elutions caused fewer peaks for performing molecular weight measurements, but became more difficult to quantify due to the increased amount of sample processed. Since bioassays are used to measure neutrophil activators, it is imperative that the stimulant be as physiological as possible in nature. In addition to computer analysis of possible neutrophil activating peptide sequences, degradation products from the two most important pancreatic serine proteases, trypsin and chymotrypsin, were clearly evaluated. The results obtained from these experiments are discussed in Example 7. Neither the cleavage of trypsinogen by trypsin or chymotrypsin, nor the cleavage of chymotrypsinogen by trypsin or chymotrypsin resulted in the formation of neutrophil activating peptides as assayed by the actin polymerization test.

The results indicate that the low molecular weight component of pancreatic homogenate responsible for neutrophil activation
Indicates that it is composed of multiple factors. Support for this is provided in part by the inability of all single inhibitors to completely control the inflammatory profile seen in neutrophil down-regulation. All pancreatic fractions containing activity, separated through the FPLC Cation MonoS column, elute in the first four fractions, indicating that the unknown pancreatic activator is uncharged or slightly cationic. Indicated. Elution through an anionic MonoQ column, which caused the separation of the elutant, also caused a separation and subsequent reduction of neutrophil activation from sample to sample. This indicates that the neutrophil activation response is essentially an adjunct to these activators (eg, the presence of a priming factor). RP-HPL
Further purification by C also caused incomplete isolation. Neutrophil activator eluted by HPLC was uniform in later fractions (# 16-17).
However, these factors represent a fraction of the original neutrophil activation response that was fractionated when they were obtained from FPLC.

A second group of potential neutrophil activators that can be produced in pancreatic homogenates are PAF-like substances. It has already been determined that no peak corresponding to the standard PAF appears in any of the mass spectra tested to date. However, it is possible that PAF-like substances can function as neutrophil activators produced by the pancreas. The mechanism of efficacy of PAF inhibitors may not be inhibition of PAF itself, but rather inhibition of neutrophil activation in response to PAF-like substances that also bind to neutrophil PAF receptors. These factors have been reported to circulate in vivo, and PAF-like substances possibly derived from cell membrane lipids such as phosphatidylcholine have been found in the plasma of smokers and smoked animals.
Their production is inhibited by antioxidants (Lehr et al. (1997) J Clin Inve.
st 99: 2358-64), indicating an oxidative mechanism in their formation.

Of the putative PAF-like substances produced either by oxidative degradation of phosphatidylcholine or by other methods, linoleate and arachidonate have about 10 μM
Stimulated the formation of neutrophil superoxide at different concentrations (Sato et al. (
1986) Physiol Chem Phys Med Nmr 18: 79-87). In contrast to similar studies by other inventors, these species were reported to lack their ability to oxidize neutrophils (Mclntyre et al. (1995) Physiology and
Oxford, Chapter 3 of the Pathophysiology of Leukocyte Activation, edited by Graner et al.
Press, Oxford, pp. 1-30). Other researchers have suggested that lysophospholipids such as lysophosphatidylcholine, or lysoPAF, and their derivatives,
It has been shown to enhance neutrophil respiratory bursts but is not reactive in nature (Smiley et al. (1991) J Biol Chem 266: 11104-11110; Lin
(1988) Scand J Clin Lab Invest 48: 303-311; Gi
(nsberg et al. (1989) Inflammation 13: 163-174; Englberger et al. (1987) International Journal of Immunopharmacy 9: 275-282).
. Related phosphocholines, such as 2-azelaoil phosphatidylcholine,
It causes cell damage and membrane lysis and can also be irritating to neutrophils (Itab.
e, et al. (1988) Biochim Biophys Acta 962: 8-15). That is, in inflammatory conditions such as ischemia, the release of lipid "priming" factors is sufficient to make cells hyperresponsive to any additional stimuli, including other phospholipids, It may be to function effectively as their own activator.

A PAF-like substance capable of activating neutrophils in vitro was found in calf brain homogenates. These products are produced by peroxidation of lipids,
It is involved in oxidative stress as a major triggering mechanism for the production of these neutrophil activators (Tokumura et al. (1987) Biochem Biophys Res Commun 145: 415-425; Tanaka et al. (1993) Biochim Biophys Acta 1166: 264- 274). The mass spectroscopy of these activators is almost exactly consistent with the mass spectroscopy performed on fraction # 2-3 of FPLC rat pancreatic homogenate. This provides compelling evidence that neutrophil activators in the pancreas can include PAF-like substances.

The close relationship between the FPLC active fraction and the mass spectrometry results of the calf PAF-like substance is not only due to the pancreatic homogenate, but also to the subsequent activation of protease-activated homogenates of other organs that respond to PAF inhibitors. A neutrophil activation assay was derived. It is known that PAF formation by the endothelium can be triggered by the serine protease thrombin (Bussolino et al. (1995) Eur J Biochem 229).
: 327-337; Carveth et al. (1992) Semin Thromb Hemost 18: 1 26-34; Zimmermann et al. (1986) Ann NY Acad Sci 485: 349.
-368). Other inventors have found that the endothelium produces PAF in response to pancreatic proteases, and that PAF production can be blocked by protease inhibitors.

In a result that shows a significant increase in neutrophil activation when neutrophils are mixed with tissue homogenates incubated with serine proteases, these mechanisms of action indicate that It appears that PAF (or a PAF-like substance) was formed in vitro. The results obtained from phospholipase C, a non-specific PAF inhibitor, indicate that neutrophil activation seen by the low molecular weight pancreatic homogenate is partially due to the fact that phospholipase C degrades PAF-like substances in addition to PAF. Is due to a similar substance. This inhibition was seen in actin polymerization and chemiluminescence assays. The use of the commercially available PAF receptor inhibitors dioxolane and WEB2170, which also competitively inhibit PAF and PAF-like factors, is relatively ineffective in reducing neutrophil activation in response to activated homogenates Prove that.

In the studies herein, neutrophil activators present in low molecular weight pancreatic homogenates were identified and partially isolated. The results obtained from FPLC, RP-HPLC and MALDI mass spectroscopy show that 3
It shows that neutrophil activators below kD are probably in the range of about 300-800D. In particular, 611, 637, 655, 673, and 6 in the filtered homogenate.
It appears to be a group of PAF-like substances with a molecular weight of 95D.

9.5 Peptides The peptides of 146 are tested programmatically to list the sequence in letters indicating the species origin of the peptide, followed by a brief description of the peptide or its possible mechanism of action (FIG. 5a- 5c). Peptide sequences were obtained from literature as well as from the 1997 Sigma Chemicals and Boehringer Mannheim chemical catalog. Most of the peptides tested are also of pancreatic origin, but other ubiquitous peptides (eg, bradykinin, fMLP) are also included. These peptides are analyzed stepwise for similarity to neutrophil activators, as well as the length of the peptide, as well as the complete peptide sequence, as well as its amino acid composition.

Example 10 Summary of Findings and Certain Conclusions Neutrophils are involved in the pathogenesis of many acute and chronic disease processes, and their improper up-regulation may lead to otherwise healthy individuals herein. Has been proposed as a risk factor for susceptibility to the disease. Plasma taken from animals after an ischemic event shows the ability to activate untreated neutrophils, and circulatory factors reduce the upregulation of neutrophils and some of the inflammation seen after these events. Indicate that you are responsible.
The presence of such activators in rat shock plasma has been identified herein. It has also been shown that among all the organs studied, only one is produced endogenously by the pancreas, which has intrinsic properties that activate neutrophils in vitro. Further experiments characterize this factor in vitro and in vivo, and many physiological properties of the pancreatic neutrophil activator have been determined. This example summarizes the findings reported in the above example.

10.1 Introduction To control neutrophil activation in vivo, the identification of key activators was first established. The major neutrophil activators in the in vivo setting appear to be able to take one of two forms: they are sufficient stimuli to fully activate neutrophils by concentration or potency Get or they are "primed"
There may be less stimulation sufficient for neutrophil activation alone. Primed neutrophils are
Cells that have been subjected to a subactivation threshold stimulus and subsequently hyperresponsive to low concentrations of activator. A large number of factors have been identified as priming agents in vitro, and this phenomenon has also been observed experimentally in vivo and clinically. In vivo neutrophil activation to a large extent is likely to depend on the priming phenomenon. In acute conditions, such as shock, there is most likely a combined synergy between previously quiescent and primed neutrophils.

Neutrophils circulate with varying degrees of activation. At any given moment, there is an activated population, a primed population and quiescent cells, and possibly circulation as non-activated marginated neutrophils. In healthy individuals, the majority of these cells are considered quiescent cells. "Pre-activation" of neutrophils is defined herein as a shift in the distribution of neutrophil populations to include a high number of primed and activated neutrophils.
This shift in neutrophil distribution was attributed to increased mortality in animals subjected to hemorrhagic and endotoxin shock, and increased post-shock lipid peroxidation levels (corresponding to early neutrophil activation). (See Example 3). The latter results include a direct role for neutrophils via oxygen free radical production which mediates the increased damage seen in pre-activated animals. Preactivation is not fully understood, but has been shown to be reduced by endotoxin resistance, probably due to a combination of factors in humans. Among other factors, factors that affect preneutrophil activation in vivo include subclinical infections, stressors and diet.

Experiments demonstrate that starved individuals have lower concentrations of plasma-loaded neutrophil activator than individuals who have recently eaten. In an autologous blood control experiment,
Lucigenin-promoted chemiluminescence has been found to be affected by consumption of previous evening meals containing significant amounts of lipids and fatty acids (our observations). Other research
(Plotnick et al. (1997) Jama 278: 1682-1686) show that high lipid consumption affects forearm blood flow vascular activity in otherwise healthy individuals. This reduction in systemic vasodilation, which is inhibited by antioxidants, can therefore occur via a free radical mechanism. This can also result in the activation or priming of neutrophil subpopulations in vivo.

10.2 Conclusions The presence of factors produced in the pancreas that lead to neutrophil activation in vivo and in vitro has been identified. Furthermore, the presence of proteases in the pancreas has been identified as a mechanism for the production of neutrophil activators in otherwise non-reactive tissues. These results indicate that the pancreas appears to be a source of proteolytic and other circulating factors that lead to neutrophil activation in shock. Limited (asymptomatic) ischemia and dietary intake can also alter the pancreatic environment, with elevated plasma levels of neutrophils "pre-activated"
That lead to increased production of proinflammatory mediators in individuals containing It is clear that the pancreas is a possible source of neutrophil activator, and if it is not controlled,
If released in large quantities in the circulation, it can have very harmful consequences. These factors appear to be transported through the thoracic duct and through the lymphatic vessels in a manner similar to MDF.

Here, the presence of a low molecular weight activator smaller than 3 kD is indicated. This factor shows a different inhibition profile than PAF. Although some inhibition of phospholipase C cleavage is shown, observation using commercial PAF factors of either BTP-dioxolane and WEB2170 showed little inhibition. Similarly, the major zymogen hydrolyzable fragments from trypsinogen and chymotrypsinogen do not tend to stimulate neutrophils, and protease inhibition of neutrophil activation is
It appears to be a neutrophil-mediated event, not an inhibition of the activator itself.

As described in Example 7, the factors released by protease incubation of non-pancreatic homogenates do not appear to be identical to those produced in the pancreas. Neutrophil activation, seen under in vivo microscopy, is associated with the interaction of the pancreatic protease with host tissues, which again form activators, as seen in vitro by incubation of the serine protease with a previously inactive homogenate. Could be the result of Most likely, neutrophil activation seen in vivo is due to a synergistic combination of the two effects. The additional synergistic effect of endogenous low molecular weight neutrophil activator and serine protease production of novel neutrophil activator is that pancreatic homogenate is a very strong stimulator of neutrophil activation, and pancreatic complications May be responsible for the shocks that occur when they occur.

In shock, the pancreas is one of the most injured organs of even limited ischemia, which can trigger the release of toxic factors into the blood. Elevated levels of circulating pancreatic proteases are routinely seen during shock, demonstrating that pancreatic factors circulate in the blood. In conditions of low etiology, different dietary conditions can lead to a limited release of neutrophil activators as seen in human plasma after ingestion of a fat diet. The concentration of pancreatic neutrophil activator appears to be sufficient to produce a systemic effect on the body.

[0440] Less than 30% of the homogenized rat pancreas is sufficient to induce lethality within minutes when injected into donor rats, and lower concentrations are equally harmful to the mind and body. In vitro, less than 1% of homogenized rat pancreas activates isolated neutrophils with greater intensity than the same volume of 1 μM PAF or fMLP. Finally, in other viscera experiments, some of the circulating neutrophil activators, which do not have an intrinsic ability to activate neutrophils and are measured systemically during shock and apparently healthy, Released from the pancreas, indicating identical.

Since modifications will be apparent to those skilled in the art, the present invention is to be construed as limited only by the appended claims.

[Brief description of the drawings]

FIG. 1. Cardiovascular cell activation plays a central role in cardiovascular disease and the immune response, and it responds to lifestyle factors and trauma, ischemia, infection; atherosclerosis Cell activation and disease, indicating that it causes poor results in trauma, shock, MI; participates in a disease-positive feedback loop; and is governed by circulating plasma factors The outline of is shown.

FIG. 2 shows cell activation diagnostic and therapeutic points (ARDS refers to adult respiratory distress syndrome, MOF refers to multiple organ failure).

FIG. 3 shows possible therapeutic intervention points. 3a) shows downstream intervention of activation, such targets being integrin IIa / IIIb for platelet aggregation, VLA-4 for T cells and eosinophils, CD-18 for neutrophil adhesion, endothelial adhesion ICAM-1, showing selections E, P for neutrophil migration; b
) Indicates pre-activation intervention by attacking the activator proposed herein.

FIG. 4 shows the chemical formulas of some proposed PAF-like factors (Itabe et a
l. (1988) Biochim Biophys Acta 145: 415-425, Englberger et al. (1987) Int
ernational J Immunopharmacy 9: 275-282; and Tanaka et al. (1993) Lab Inve
st 70: 684-695). The last set of PAF-like factors with variable sn-2-side chains is from bovine brain and may be identical to the activators found in pancreatic homogenates provided herein.

Figures 5a-5c show a list of peptides tested with the computer programs described herein. The latter indicates the origin of the species of the peptide, followed by a brief description of the peptide, ie its possible mechanism of action. Legend of peptide origin: b = bovine; h = hamster; m = human; o = others; r = rat. These peptides are
The pancreatic homogenates provided herein were compared in size to those found by mass spectrometry.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] June 2, 2000 (2006.2)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 9/00 A61P 9/10 9/10 25/28 25/28 27/02 27/02 37/02 37 / 02 37/06 37/06 43/00 111 43/00 111 C12Q 1/02 C12Q 1/02 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 C12N 5/00 E // A61K 38 / 55 A61K 37/64 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, G, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE , ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG , US, UZ, VN, YU, ZW (71) Applicant The Regents of the University of California THE REGENTS OF THE UNIVERSITY OF CALIF ORNIA United States of America, California 94607-5200, Auckland, Franklin Street 1111, Twelve's Floor (71) Applicant The Scripps Research Institute USA California 92037 La Jolla North Torrey Pines Road 10550 (72) Inventor Roland Bee Stoughton United States 92103 West Spruce Street, No. 435, San Diego, California Inventor Geert B. Schmidt-Schombingen 92014 No. 13425, Mango, Del Mar, California (72) Inventor Tony E. Fooglie, United States 92122 No. 6026, Chalet Street, San Diego, California (72) Inventor Eric Kisler 92122, Avenida Navidad Number 109, 7936, San Diego, CA

Claims (65)

[Claims] 1. A method of producing a cell activating composition, comprising: homogenizing pancreatic tissue in a buffer at approximately neutral or higher pH to produce a homogenate; removing particles from the homogenate. Optionally incubating the resulting homogenate with the removed particles and a protease; fractionating the homogenate and selecting a fraction exhibiting cell activating activity. 2. The process according to claim 1, wherein the homogenate is fractionated by size; and the molecular weight in the homogenate is reduced to 3 kD and / or a component exceeding 3 kD, whereby the fractionation is performed. 3. The method of claim 1, further comprising subjecting the resulting homogenate to reverse phase medium pressure liquid chromatography; and selecting and combining fractions having cell activating activity. 3. The method according to 2. 4. The method according to claim 1, further comprising subjecting the resulting active fraction to reverse phase chromatography; and selecting and combining a fraction having cell activating activity. The method described in Crab. 5. Assessing cell activating activity by measuring neutrophil free radical formation, pseudopodia formation, adhesion molecule expression, granule release, inflammatory mediator production, or some combination thereof. A method according to any of claims 1-4. 6. A cell activating composition produced by the method according to claim 1. 7. A method of improving treatment outcome or reducing the risk of treatment, comprising assessing the treatment option for a disease or condition by measuring the level of cell activation in a subject; Administering a therapy that reduces activation, and then initiating further treatment of the disease or condition. 8. The method of claim 7, wherein the treatment is a veterinary treatment of a non-human subject. 9. The method of claim 7, wherein the cell activation is assessed by an assay that measures one or more of neutrophil free radical production, pseudopodia formation, adhesion molecule expression and degranulation. The method described in. 10. The disease or condition to be treated is cardiovascular disease, inflammatory disease, trauma,
Autoimmune diseases, arthritis, organ rejection, diabetes and diabetic complications, seizures, ischemia,
10. The method according to any one of claims 7-9, wherein the method is selected from Alzheimer's disease. 11. The method according to claim 7, wherein the disorder is selected from the group consisting of myocardial infarction, stroke, hemorrhagic shock, diabetic retinopathy, diabetes and venous insufficiency. 12. The method according to any of claims 7 to 11, wherein the procedure to be evaluated is a surgery, a procedure for unstable angina or a procedure for trauma. 13. The method of any of claims 7 to 12, wherein the treatment that reduces activation comprises administering a protease inhibitor, dialysis, changing lifestyle to reduce stress, or changing diet. The method described in. 14. The protease inhibitor is a serine protease inhibitor.
A method according to any of claims 7-13. 15. The protease inhibitor is selected from α ₁ -proteinase inhibitor (α ₁ -antitrypsin), α ₂ -macroglobin, inter-α ₁ -trypsin inhibitor, and α ₁ -antichymotrypsin. 15. The method according to any of claims 7-14. 16. The method according to any one of claims 7-14, wherein the protease inhibitor is 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate or a pharmaceutically acceptable salt, acid, ester and other derivatives thereof. The method described in Crab. 17. Use of a protease inhibitor for the formulation of a drug that reduces cell activation. 18. Use of a protease inhibitor to reduce cell activation. 19. The enhanced cell activation of cardiovascular disease, inflammatory disease, trauma, autoimmune disease, arthritis, diabetes and diabetic complications, seizures, ischemia, Alzheimer's disease, or increased cell activation. Use according to 18. 20. The use according to claim 17 or claim 18, wherein enhanced cell activation results from myocardial infarction, stroke, hemorrhagic shock, diabetic retinopathy, diabetes and venous insufficiency. 21. The protease inhibitor is a serine protease inhibitor,
Use according to any of claims 17-20. 22. The protease inhibitor is 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate, a chymotrypsin or trypsin inhibitor, or a pharmaceutically acceptable salt, acid, ester and other derivatives thereof. 21. Use according to any of claims 17-20. 23. protease inhibitor is alpha ₁ - proteinase inhibitor (alpha ₁ - antitrypsin), alpha ₂ - macro globin, inter-.alpha. ₁ - trypsin inhibitor, Contact and alpha ₁ - a antichymotrypsin, claim Use according to any of 17-20. 24. The method of any one of claims 17 to 20, wherein the protease inhibitor is 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate or a pharmaceutically acceptable salt, acid, ester and other derivatives thereof. Use as described in Crab. 25. A method for treating or preventing a disorder mediated by inappropriate cell activation, comprising administering an effective amount of a protease inhibitor, the amount effective to reduce cell activation. The method comprising. 26. The method according to claim 25, wherein the protease is a serine protease inhibitor. 27. The protease inhibitor is a 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate, chymotrypsin or trypsin inhibitor, or a pharmaceutically acceptable salt, acid, ester and other derivatives thereof. 26. The method of claim 25. 28. The protease inhibitor is an α ₁ -proteinase inhibitor (α ₁ -antitrypsin), α ₂ -macroglobin, an inter-α ₁ -trypsin inhibitor, and an α ₁ -antichymotrypsin. 25. The method according to 25. 29. The method of claim 25, wherein the protease inhibitor is 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate or a pharmaceutically acceptable salt, acid, ester and other derivatives thereof. . 30. The method of claim 25, wherein the disorder is selected from the group consisting of myocardial infarction, stroke, hemorrhagic shock, diabetic retinopathy, diabetes and venous insufficiency. 31. A product comprising a pharmaceutical composition comprising a packaging material and a protease inhibitor contained within the packaging material, wherein the pharmaceutical composition reduces cell activation or cellular activity. A product which is effective in preventing an increase in cell activation, wherein the packaging material includes a label indicating that the pharmaceutical composition is used to reduce the level of cell activation. 32. The protease inhibitor is a serine protease inhibitor.
The product of claim 31. 33. protease inhibitor is alpha ₁ - proteinase inhibitor (alpha ₁ - antitrypsin), alpha ₂ - macro globin, inter-.alpha. ₁ - trypsin inhibitor, Contact and alpha ₁ - it is selected from among antichymotrypsin 32. The product of claim 31, wherein the product. 34. The product of claim 31, wherein the protease inhibitor is 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate or a pharmaceutically acceptable salt, acid, ester and other derivatives thereof. . 35. A method for identifying a compound that reduces the level of cell activation, comprising contacting a cultured cell with the composition of claim 5, or with any known cell activator and test compound. Contacting simultaneously or sequentially; measuring the level of cell activation in the cell; and selecting a compound that inhibits the cell activating activity of the composition. 36. The method of claim 35, wherein the cells are endothelial cells. 37. The method of claim 35 or claim 36, wherein after contacting the cell with the composition, the cell is contacted with the compound. 38. A method of diagnosis and treatment comprising assessing cell activation; and, if so, administering a therapy that reduces activation. 39. The method of claim 38, wherein the treatment that reduces activation comprises changing diet and / or lifestyle. 40. The method of claim 38 or claim 39, wherein the treatment that reduces activation comprises administration of a protease inhibitor. 41. The protease inhibitor is a serine protease inhibitor.
41. The method according to claim 40. 42. A protease inhibitor is alpha ₁ - proteinase inhibitor (alpha ₁ - antitrypsin), alpha ₂ - macro globin, inter-.alpha. ₁ - trypsin inhibitor, Contact and alpha ₁ - is selected from among antichymotrypsin 41. The method of claim 40, wherein 43. The method of claim 38, wherein the therapy that reduces activation comprises dialysis or extracorporeal blood treatment. 44. A method of measuring cell activation in a subject, comprising contacting quiescent cultured cells with plasma obtained from the subject; and detecting activation of the cultured cells. . 45. A method of screening a population of patients for a drug clinical trial comprising: testing the level of cell activation in each member of the population; identifying a member having a high level of cell activation; Select patients for testing or exclude them from the study, where the cell activation level is low if insufficient results due to such high levels hinder the evaluation of test results Excluding patients with high cell activation; if the test assesses the effect on cell activation, patients with high cell activation levels are included. 46. The method of claim 45, wherein the high level is a level predetermined to be statistically significant. 47. The method of claim 45, wherein the high level is about 10% above the average or some standard deviation level. 48. A kit for diagnosing and treating a disorder associated with cell activation, the kit comprising: a method for evaluating cell activation; and a method for reducing cell activation. 49. The kit of claim 48, wherein the method of assessing cell activation comprises a method of assessing superoxide production and / or actin polymerization. 50. The method of reducing cell activation, wherein the method comprises a protease inhibitor, aspirin, propranolol, heparin or coumarin.
50. The kit according to 49. 50. A kit comprising: a method for measuring hydrogen peroxide; and a composition comprising superoxide dismutase. 51. The kit of claim 50, further comprising a composition comprising catalase. 52. The kit according to claim 50, further comprising a microtiter plate and a reagent for measuring hydrogen peroxide. 53. The kit of claim 53, wherein the reagent for measuring hydrogen peroxide is in a well of a microtiter plate. 54. The kit of claim 50, wherein the method for measuring hydrogen peroxide comprises a chromogen and an oxidizing agent. 55. The method for measuring hydrogen peroxide comprising an electrode.
The kit according to any of 0-52. 56. The kit of any of claims 50-53, wherein the chromogen is phenol red and the oxidizing agent is horseradish peroxidase. 57. A method for assessing the level of reactive oxygen species (ROS) in a plasma or blood sample, comprising: (a) obtaining a plasma or whole blood sample and measuring hydrogen peroxide levels; C.) Measuring peroxide levels in the presence of superoxide dismutase (SOD); and (c) comparing the results obtained from the first measurement with the results obtained in the presence of SOD, thereby reducing Measuring the amount of ROS produced in the method. 58. A comparison of each measurement with a control; except that each control further comprises catalase for converting all hydrogen peroxide to water and oxygen, the control comprises a sample (a 58.) and sample (b). 59. (a) adding a plasma or whole blood sample to a mixture of a chromogen reagent and an oxidant reagent, or adding multiple samples to the wells of a plate comprising these reagents, (B) adding SOD to the mixture or each well and measuring the change in optical density in the mixture or each well; and (c) adding catalase to the mixture or each well and adding The optical density (OD) in the wells was measured, where the measurement after the addition of catalase was a control; the comparison between measurements (a) and (c) showed that the hydrogen peroxide produced by the sample was 58. The method of claim 57, comprising: providing a quantity; (a) and (b) providing a measure of the amount of ROS in the sample. 60. The method of any of claims 57-59, wherein an elevated level of hydrogen peroxide in the measurement of (a) compared to a control sample (c) indicates the presence of an inflammatory condition. 61. The method of any of claims 57-59, wherein the first (a) and second (b) sample measurements indicate leukocyte up-regulation. 62. The method of claim 61, wherein said up-regulation is indicative of the onset of an acute cardiovascular disorder, such as disease onset or ischemic complication. 63. The method of claim 61, wherein the up-regulation is indicative of an ischemic complication. 64. An enhanced first (a) measurement and a lower second sample (
60. The method of any of claims 57-59, wherein the measurement of b) indicates the presence of a chronic or immunocompromised condition. 65. The chronic or immunocompromised condition is hypertension or sepsis.
65. The method of claim 64.